副粘病毒F蛋白HR1和HR2在特异性膜融合中的作用

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NDVC-HR1和hPIVC-HR1。用同样的方法又得到交换HR2的两个嵌合体。即NDVC-HR2和hPIVC-HR2。将各种嵌合体DNA与同源及异源HNDNA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，NDVC-HR1的融合功能只有野毒株的53．91％，hPIVC-HR1的融合功能达到野毒株的83．15％；交换HR2后，NDVC-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NDVC-HR1和hPIVC-HR1的表达效率均下降。交换HR2后，NDVC-HR2的表达效率没有改变，而hPIVC-HR2的表达效率下降。结论NDVF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；hPIVF的HR1和HR2对hPIV的特异性膜融合都有重要作用。

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

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 eukaryocytes.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 proteins 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.