副黏病毒F蛋白融合活性位点中病毒特异性氨基酸基因突变分析

目的 了解副黏病毒融合蛋白(F)融合活性位点中的病毒特异性氨基酸在细胞融合中的作用.方法 以新城疫病毒(NDV)和人副流感病毒(hPIV)为例,在已确定的F蛋白融合活性位点中对病毒特异性氨基酸进行定点突变,然后将突变体F基因与同源或异源的血凝素.神经氨酸酶(HN)基因共转染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 在NDV F的突变体中,N150D-L152D的融合功能达到野毒株的46.31%;而N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E的融合活性却几乎消失,分别只有野毒株的1.25%、3.14%和2.23%;N296D-N297D的融合功能是野毒株的97.68%.在hPIV F的突变体中,D143A-E145A的融合功能达到野毒株的32.63%;E223Q-K224A几乎不能形成合胞体,其融合活性只有野毒株的1.91%;K263A-R265A、D268A-D270A和R475A-R476A的融合功能分别是野毒株的14.63%、19.52%和28.95%.FACS结果表明,NDV F的N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E突变体及hPIVF的E223Q-K224A突变体F蛋白在细胞表面几乎没有表达;其余所有突变体F蛋白的表达效率与野毒株相比,基本不变.结论 对于NDV F来说,N257、N258、Q259、G271、N272、Q279、Q281对NDV F的特异性细胞融合功能起重要作用;N150和L152也起一定的作用,但是N296和N297却没有作用.对于hPIV F来说,E223和K224对hPIV F的特异性细胞融合功能起非常重要的作用;D143、E145、K263、R265、D268、D270、R475、R476的作用也很重要.

Mutational analysis of virus specific amino acids in the fusion active domain of paramyxovirus fusion protein

Objective To identify the effects of virus specific amino acids in the fusion active domains of paramyxovirus fusion proteins on the specific membrane fusion. Methods Site-directed mutagenesis was used to obtain mutants in the identified fusion active domains of Newcastle disease virus (NDV) fusion protein (F) and human parainfluenza virus (hPIV) fusion protein (F). All the mutant F genes were co-expressed with their homol-ogous or heterogenous hemagglutinin-neuraminidase (HN) genes in eukaryocytes. The fusion functions of mutants were assayed by Giemsa staining and reporter gene method. The expression efficiencies of mutants were assayed by fluorescence-activated cell sorter (FACS). Results In the NDV F mutants, N150D-L152D had 46.31% fusion activity of wide type. The fusion activities of N257D-N258D-Q259E, G271D-N272D and Q279E-Q281E almost disappeared, and they had only 1.25%, 3.14% and 2.23% of fusion activities, respectively, compared with wide type. N296D-N297D had 97.68% fusion activity of wide type. In the hPIV F mutants, D143A-E145A had 32.63% fusion activity of wide type. The fusion activity of E223Q-K224A almost disappeared, and it had only 1.91% fusion activity of wide type. K263A-R265A, D268A-D270A and R475A-R476A had 14.63%, 19.52% and 28.95% of fusion activities respectively compared with wild type. The analysis of FACS indicated that proteins of NDV F N257D-N258D-Q259E, G271D-N272D, Q279E-Q281E and hPIV F E223Q-K224A were not expressed on the cell surface, while proteins of the rest mutants were expressed nearly as the same as the wide types. Con-clusion As to NDV F, the amino acids of N257, N258, Q259, G271, N272, Q279 and Q281 were significant to the specific membrane fusion, and N150 and L152 were also important, but N296 and N297 were not. For hPIV F, the amino acids of E223 and K224 were significant to the specific membrane fusion, and D143, E145, K263, 11265, D268, D270, R475 and R476 were also important.