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作 者:刘晶雪 刘颖[1] 迟苗苗 姜晶晶 操詹魁 温红玲[1] 赵丽[1] 迟连利[2] 王志玉[1] LIU Jingxue;LIU Ying;CHI Miaomiao;JIANG Jingjing;CAO Zhankui;WEN Hongling;ZHAO Li;CHI Lianli;WANG Zhiyu(Department of Virology, School of Public Health, Shandong University, Jinan 250012, China;National Gl ycoengineering Research Center, State Key Laboratory of Microbial Technology, Shandong University, J inan 250100, China)
机构地区:[1]山东大学公共卫生学院、病毒学研究室,济南250012 [2]山东大学国家糖工程技术研究中心微生物技术国家重点实验室,济南250100
出 处:《病毒学报》2018年第3期318-327,共10页Chinese Journal of Virology
基 金:国家自然科学基金(项目号:81672011),题目:副粘病毒HN蛋白颈部与自身头部、F蛋白的相互作用及机制;山东大学交叉培育项目(项目号:2015JC044),题目:病毒感染性疾病相关糖链的结构与功能研究~~
摘 要:副粘病毒(Paramyxovirus)包膜上镶嵌着两种糖蛋白血凝素-神经氨酸酶(Hemagglutinin-neuraminidase,HN)和融合蛋白(Fusion protein,F),两者的相互作用是决定病毒宿主范围、毒力和传播的关键。为探讨HN颈部与F相互作用区(Fusion interaction region,FIR)在膜融合机制中的作用,选取新城疫病毒(Newcastle disease virus,NDV)与人副流感病毒3型(human parainfluenza virus type 3,hPIV3)为研究对象,通过片段置换及同源重组技术构建嵌合体C1、C2,进一步将NDV及hPIV3HN的FIR内第51位丝氨酸(Serine,S)、第55位天冬氨酸(Aspartic acid,D)定点突变为丙氨酸(Alanine,A),获得突变体NDV S51A、NDV D55A、hPIV3S51A、hPIV3D55A,对嵌合体及突变体蛋白的细胞表面表达效率、受体识别活性、神经氨酸酶活性、促细胞融合活性及半融合活性进行检测。结果:各嵌合体C1、C2及突变体NDV S51A、NDV D55A、hPIV3S51A、hPIV3D55A的细胞表达效率、神经氨酸酶活性(Neuraminidase,NA)与野生型相比差异不显著(P〉0.05),但促细胞融合活性均有不同程度的降低(P〈0.05),C1、C2、NDV S51A、NDV D55A、hPIV3S51A、hPIV3D55A分别为野生型的7%、9%、27%、19%、17%和21%;C1、C2、NDV S51A、NDV D55A、hPIV3S51A、hPIV3D55A的受体识别活性分别为14.7%、22.3%、35.5%、28.8%、33.9%和40.2%,与野生型相比差异显著(P〈0.05)。结果表明:副粘病毒HN蛋白颈部与F相互作用区的突变及置换使HN蛋白的促细胞融合活性、受体识别活性降低,其中第51位丝氨酸(S51)及第55位天冬氨酸(D55)发挥重要作用。In paramyxoviruses,the interactions between fusion protein(F)and hemagglutinin-neuraminidase(HN)are critical in determining the host range,virulence and spread of these viruses.To investigate the function of the fusion interaction region(FIR)in the mechanism of membrane fusion,Newcastle disease virus(NDV)and human parainfluenza virus type 3(hPIV3)were selected as research objects.Site-directed and fragment replacement mutagenesis methods were used to obtain chimeras,then we identified two conserved amino acids,serine(S)and aspartic acid(D)in the FIR through comparison with highly similar sequences in NDV and hPIV3 HN,and mutated them into alanine respectively,namely NDV S51 A,NDV D55 A,hPIV3 S51 Aand hPIV3 D55 A.Cell surface expression,as well as the activity of receptor binding,neuraminidase,fusion promotion and semi-fusion,was determined.There was no significant difference in cell surface expression or neuraminidase activity between each chimera,mutant and wildtype(P〈0.05),but fusion promotion activity was reduced significantly(P〈0.05):C1,C2,NDV S51 A,NDV D55 A,hPIV3 S51 A,and hPIV3 D55 A was 7%,9%,27%,19%,17% and 21% of the wild-type level,respectively.C1,C2,NDV S51 A,NDV D55 A,hPIV3 S51 Aand hPIV3 D55 Aalso showed reductions in receptor binding activity,which was 14.7%,22.3%,35.5%,28.8%,33.9% and 40.2%,respectively.These data suggest that the FIR in the stalk domain plays a very important role in the fusion promotion activity and receptor binding activity of NDV and hPIV3.S51 and D55 are key amino acids in the FIR in the stalk domain.
关 键 词:副粘病毒(Paramyxovirus) 血凝素神经氨酸酶(HN) 融合蛋白(F) 相互作用区
分 类 号:R373.9[医药卫生—病原生物学]
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