NA蛋白对流感病毒受体结合特性影响的研究
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摘要
为研究两株H7亚型流感病毒A/chicken/Jilin/SD020/2014(H7N2)(简称JL/020)和A/Anhui/1/2013(H7N9)(简称AH/1)受体结合特异性差异的影响机制,本实验利用反向遗传操作技术,构建一系列重配病毒和HA基因点突变病毒,检测其对受体结合特性的影响。固相ELISA检测结果表明血凝素蛋白(HA)中的57和312位氨基酸不影响流感病毒的受体结合特性,而神经氨酸酶蛋白(NA)使r-JL/020(AH/1骨架)结合SAα2,3Gal受体的结合能力高于r-AH/1(R57K/R312K),表明NA影响了流感病毒受体结合特性;同源建模进一步发现HA中的57和312位点与流感病毒受体结合结构域相距较远;交叉血凝抑制试验(HI)结果表明,H7单因子血清和H7N9全病毒血清对拯救的JL/020和AH/1病毒株的抑制价没有差异,而N2单因子血清对病毒的抑制能力存在差异。以上结果表明,NA蛋白影响了流感病毒的受体结合特性。本研究表明,除HA以外,NA也能够影响流感病毒的受体结合特性,该实验为进一步研究流感病毒受体结合特性提供了实验依据。
Two H7 subtype influenza viruses, A/chicken/Jilin/SD020/2014(H7N2)(JL/020) and A/Anhui/1/2013(H7N9)(AH/1), were selected to study the mechanism of receptor-binding specificity in this study. To investigate which factor influences the receptor-binding property, a series of reassortants and mutant viruses were constructed by using 8 plasmid-based reverse genetics. Solid-phase ELISA assay showed that the receptor-binding specificity of AH/1 was not affected by the R57 K and R312 K mutations in HA, while N2 NA increased the binding affinity against SAα2,3Gal of r-JL/020(AH/1 backbone), indicating that NA influenced the receptor-binding property. Homologous modeling further showed that the aa57 and aa312 in HA were not located in the receptor-binding domains. The hemagglutination inhibition(HI) antibody titers of the H7 HA single factor and H7N9 whole virus sera against JL/020 and AH/1 virus were both 4 and 7, respectively, while the HI titers of the N2 NA sera against the two viruses were different. All these results indicated that NA protein influenced the receptor-binding properties. This study demonstrates that, in addition to HA, NA has also an important role on receptor-binding specificity of influenza A virus, which provides a basis for further study of receptor-binding properties.
Two H7 subtype influenza viruses, A/chicken/Jilin/SD020/2014(H7N2)(JL/020) and A/Anhui/1/2013(H7N9)(AH/1), were selected to study the mechanism of receptor-binding specificity in this study. To investigate which factor influences the receptor-binding property, a series of reassortants and mutant viruses were constructed by using 8 plasmid-based reverse genetics. Solid-phase ELISA assay showed that the receptor-binding specificity of AH/1 was not affected by the R57 K and R312 K mutations in HA, while N2 NA increased the binding affinity against SAα2,3Gal of r-JL/020(AH/1 backbone), indicating that NA influenced the receptor-binding property. Homologous modeling further showed that the aa57 and aa312 in HA were not located in the receptor-binding domains. The hemagglutination inhibition(HI) antibody titers of the H7 HA single factor and H7N9 whole virus sera against JL/020 and AH/1 virus were both 4 and 7, respectively, while the HI titers of the N2 NA sera against the two viruses were different. All these results indicated that NA protein influenced the receptor-binding properties. This study demonstrates that, in addition to HA, NA has also an important role on receptor-binding specificity of influenza A virus, which provides a basis for further study of receptor-binding properties.
引文
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[3]Zhang qian-yi,Shi jian-zhong,Deng guo-hua,et al.H7N9 influenza viruses are transmissible in Ferrets by respiratory droplet[J].Science,2013,341(6144):410-414.
[4]Schrauwen E J,Fouchier R A.Host adaptation and transmission of influenza A viruses in mammals[J].E merg Microbes Infect,2014,3(2):9-12.
[5]Xu Rui,Zhu Xue-yong,Ryan M,et al.Functional balance of the hemagglutinin and neuraminidase activities accompanies the emergence of the 2009 H1N1 influenza pandemic[J].Rev Med Virol,2012,12(3):159-166.
[6]Shi Jian-zhong,Deng Guo-hua,Zeng Xian-ying,et al.Novel influenza A(H7N2)virus in chickens,Jilin province,China,2014[J].Emerg Infect Dis,2014,20(10):1719-1722.
[7]李媛媛,郭晶,李旭勇,等.H7N9亚型禽流感病毒反向遗传操作系统的建立及其疫苗候选株的构建[J].中国预防兽医学报,2014,36(5):407-409.
[8]De Graaf M,Fouchier R A.Role of receptor binding specificity in influenza A virus transmission and pathogenesis[J].EMBOJ,2014,33(8):823-841.
[9]Zhu xue-yong,McB ride R,Nycholat C M,et al.Influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic acid receptors[J].J Virol,2012,86(24):13371-13383.
[10]Dortmans J C,Dekkers J,Wickramasinghe I N,et al.Adaptation of novel H7N9 influenza A virus to human receptors[J].Sci Rep,2013,3:3058-3063.
[11]Air G M,Laver W G.Red cells bound to influenza virus N9neuraminidase are not released by the N9 neuraminidase activity[J].Virology,1995,211(1):278-284.
[12]Varghese J N,Colman P M,Avan Donkelaar,et al.Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases[J].Proc Natl Acad Sci USA,1997,94(22):11808-11812.