中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2009年
6期
485-490
,共6页
柴相君%任桂芳%潘新良%任桂杰%王志玉
柴相君%任桂芳%潘新良%任桂傑%王誌玉
시상군%임계방%반신량%임계걸%왕지옥
副黏病毒%融合蛋白%活性位点%病毒特异性氨基酸%基因突变
副黏病毒%融閤蛋白%活性位點%病毒特異性氨基痠%基因突變
부점병독%융합단백%활성위점%병독특이성안기산%기인돌변
Paramyxovirus%Fusion protein%Active domain%Virus specific amino acids%Gene mutagen-esis
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.
目的 瞭解副黏病毒融閤蛋白(F)融閤活性位點中的病毒特異性氨基痠在細胞融閤中的作用.方法 以新城疫病毒(NDV)和人副流感病毒(hPIV)為例,在已確定的F蛋白融閤活性位點中對病毒特異性氨基痠進行定點突變,然後將突變體F基因與同源或異源的血凝素.神經氨痠酶(HN)基因共轉染BHK21細胞後,在真覈細胞中錶達.Giemsa染色和指示基因法檢測細胞融閤功能,熒光彊度分析(FACS)檢測F蛋白的錶達效率.結果 在NDV F的突變體中,N150D-L152D的融閤功能達到野毒株的46.31%;而N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E的融閤活性卻幾乎消失,分彆隻有野毒株的1.25%、3.14%和2.23%;N296D-N297D的融閤功能是野毒株的97.68%.在hPIV F的突變體中,D143A-E145A的融閤功能達到野毒株的32.63%;E223Q-K224A幾乎不能形成閤胞體,其融閤活性隻有野毒株的1.91%;K263A-R265A、D268A-D270A和R475A-R476A的融閤功能分彆是野毒株的14.63%、19.52%和28.95%.FACS結果錶明,NDV F的N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E突變體及hPIVF的E223Q-K224A突變體F蛋白在細胞錶麵幾乎沒有錶達;其餘所有突變體F蛋白的錶達效率與野毒株相比,基本不變.結論 對于NDV F來說,N257、N258、Q259、G271、N272、Q279、Q281對NDV F的特異性細胞融閤功能起重要作用;N150和L152也起一定的作用,但是N296和N297卻沒有作用.對于hPIV F來說,E223和K224對hPIV F的特異性細胞融閤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.
목적 료해부점병독융합단백(F)융합활성위점중적병독특이성안기산재세포융합중적작용.방법 이신성역병독(NDV)화인부류감병독(hPIV)위례,재이학정적F단백융합활성위점중대병독특이성안기산진행정점돌변,연후장돌변체F기인여동원혹이원적혈응소.신경안산매(HN)기인공전염BHK21세포후,재진핵세포중표체.Giemsa염색화지시기인법검측세포융합공능,형광강도분석(FACS)검측F단백적표체효솔.결과 재NDV F적돌변체중,N150D-L152D적융합공능체도야독주적46.31%;이N257D-N258D-Q259E、G271D-N272D화Q279E-Q281E적융합활성각궤호소실,분별지유야독주적1.25%、3.14%화2.23%;N296D-N297D적융합공능시야독주적97.68%.재hPIV F적돌변체중,D143A-E145A적융합공능체도야독주적32.63%;E223Q-K224A궤호불능형성합포체,기융합활성지유야독주적1.91%;K263A-R265A、D268A-D270A화R475A-R476A적융합공능분별시야독주적14.63%、19.52%화28.95%.FACS결과표명,NDV F적N257D-N258D-Q259E、G271D-N272D화Q279E-Q281E돌변체급hPIVF적E223Q-K224A돌변체F단백재세포표면궤호몰유표체;기여소유돌변체F단백적표체효솔여야독주상비,기본불변.결론 대우NDV F래설,N257、N258、Q259、G271、N272、Q279、Q281대NDV F적특이성세포융합공능기중요작용;N150화L152야기일정적작용,단시N296화N297각몰유작용.대우hPIV F래설,E223화K224대hPIV F적특이성세포융합공능기비상중요적작용;D143、E145、K263、R265、D268、D270、R475、R476적작용야흔중요.
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.
