Attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site
Attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site
复制标题
DOI：
10.1128/jvi.74.20.9601-9609.2000
复制
发表时间：
2000-10-01
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;Allison, SL;Heinz, FX
用定点突变的方法研究了森林脑炎病毒囊膜蛋白E的特定区域对该病毒生物学的影响。根据TEE病毒蛋白E的X射线结构，详细分析了4个氨基酸残基(E308到E311)，它们位于结构域III的上外侧表面，是黄病毒潜在的受体结合决定簇的一部分。构建了含有单一氨基酸替代的突变体，并分析了它们对小鼠的毒力、培养细胞的生长特性和遗传稳定性。小鼠体内最显著的减毒作用是苏氨酸310的诱变。将该突变与3‘-非编码区的缺失突变相结合，产生了高度减弱的突变，但突变Thr 310到Lys的生物学效应可以在很大程度上被感染小鼠时自发产生的邻近位置的突变(Lys 311到Glu)逆转，其他位置的突变为残基308(Asp)的功能重要性及其与残基311(Lys)的电荷相互作用提供了证据，而残基309可以被改变甚至删除，而不会产生任何显著的后果。伴随着残基309的缺失的是位于332位的自发第二位点突变(Phe到Tyr)，在蛋白质E的三维结构中，该突变在空间上接近残基309。这项研究中获得的信息对于开发可能作为未来活疫苗的特定减毒黄病毒株是相关的。
The impact of a specific region of the envelope protein E of tick-borne encephalitis (TBE) virus on the biology of this virus was investigated by a site-directed mutagenesis approach. The four amino acid residues that were analyzed in detail (E308 to E311) are located on the upper-lateral surface of domain III according to the X-ray structure of the TEE virus protein E and are part of an area that is considered to be a potential receptor binding determinant of flaviviruses, Mutants containing single amino acid substitutions, as well as combinations of mutations, were constructed and analyzed for their virulence in mice, growth properties in cultured cells, and genetic stability. The most significant attenuation in mice was achieved by mutagenesis of threonine 310. Combining this mutation with deletion mutations in the 3'-noncoding region yielded mutants that were highly attenuated, The biological effects of mutation Thr 310 to Lys, however, could be reversed to a large degree by a mutation at a neighboring position (Lys 311 to Glu) that arose spontaneously during infection of a mouse, Mutagenesis of the other positions provided evidence for the functional importance of residue 308 (Asp) and its charge interaction with residue 311 (Lys), whereas residue 309 could be altered or even deleted without any notable consequences. Deletion of residue 309 was accompanied by a spontaneous second-site mutation (Phe to Tyr) at position 332, which in the three-dimensional structure of protein E is spatially close to residue 309. The information obtained in this study is relevant for the development of specific attenuated flavivirus strains that may serve as future live vaccines.