Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo
Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo
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DOI：
10.1128/jvi.75.12.5627-5637.2001
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发表时间：
2001-06-01
影响因子：
5.4
通讯作者：
Heinz, FX
中科院分区：
文献类型：
--
作者：
Mandl, CW;Kroschewski, H;Heinz, FX
黄病毒蜱传脑炎病毒在BHK-21细胞中的繁殖选择大的表面糖蛋白E内的突变，增加了蛋白质的净正电荷。在16个独立实验的过程中，鉴定了12种不同的蛋白E突变模式。这些突变位于所有三个结构域中，并且分布在蛋白E的几乎整个上表面和侧表面上，这些突变导致形成主要为正表面电荷的局部斑块。在确定的遗传骨架中携带这些突变中的一些的重组病毒显示硫酸乙酰肝素（HS）依赖性表型，导致对BHK-21细胞的特异性感染性和结合亲和力增加，在猪肾细胞中形成小斑块，并且在成年小鼠中神经侵袭性显著减弱。我们的研究结果证实了这样的概念，即选择减毒HS结合突变体是一种常见的和频繁的现象，在细胞培养中的病毒繁殖，并建议在黄病毒减毒HS依赖的主要作用。认识到这一原理对今后黄病毒减毒株的设计具有一定的实用价值。
Propagation of the flavivirus tick-borne encephalitis virus in BHK-21 cells selected for mutations within the large surface glycoprotein E that increased the net positive charge of the protein. In the course of 16 independent experiments, 12 different protein E mutation patterns were identified. These were located in all three of the structural domains and distributed over almost the entire upper and lateral surface of protein E, The mutations resulted in the formation of local patches of predominantly positive surface charge. Recombinant viruses carrying some of these mutations in a defined genetic backbone showed heparan sulfate (HS)dependent phenotypes, resulting in an increased specific infectivity and binding affinity for BHK-21 cells, small plaque formation in porcine kidney cells, and significant attenuation of neuroinvasiveness in adult mice. Our results corroborate the notion that the selection of attenuated HS binding mutants is a common and frequent phenomenon during the propagation of viruses in cell culture and suggest a major role for HS dependence in flavivirus attenuation. Recognition of this principle may be of practical value for designing attenuated flavivirus strains in the future.