Spontaneous mutations restore the viability of Tick-borne encephalitis virus mutants with large deletions in protein C
Spontaneous mutations restore the viability of Tick-borne encephalitis virus mutants with large deletions in protein C
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DOI：
10.1128/jvi.77.1.443-451.2003
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发表时间：
2003-01-01
影响因子：
5.4
通讯作者：
Mandl, CW
中科院分区：
文献类型：
--
作者：
Kofler, RM;Leitner, A;Mandl, CW
森林脑炎病毒衣壳蛋白C最近被发现可以耐受长达16个氨基酸残基的缺失，这些氨基酸残基部分去除了中央疏水结构域，这是黄病毒中保守的序列元件，可能对病毒粒子组装至关重要。在本研究中，缺失长度为19、21、27或30个残基的突变体被发现移除了更多或全部这个疏水结构域，从而产生了存活的病毒后代，但这无一例外地伴随着蛋白C中额外突变的出现。这些点突变或序列重复位于工程缺失的下游，通常增加了疏水性，表明它们可能补偿了中心疏水结构域的丢失。其中两个第二位点突变与相应的缺失一起被引入到野生型遗传骨干中，对这些“双突变体”的分析提供了直接证据，证明缺失突变体的生存能力确实取决于第二位点突变的存在。我们的结果证实了蛋白C的疏水相互作用对于传染性黄病毒颗粒的组装是必不可少的，但排除了中央疏水结构域的单个残基对于传染性是绝对必要的可能性。此外，与先前描述的16个氨基酸残基缺失突变体相比，这两个突变体被发现具有高度的减毒作用，并能够在小鼠中诱导保护性免疫反应，这表明大片段缺失和第二位点突变的组合可能是产生安全、减毒黄病毒疫苗株的一种更好的方法。
The capsid protein, C, of tick-borne encephalitis virus has recently been found to tolerate deletions up to a length of 16 amino acid residues that partially removed the central hydrophobic domain, a sequence element conserved among flaviviruses which may be crucial for virion assembly. In this study, mutants with deletion lengths of 19, 21, 27, or 30 residues, removing more or all of this hydrophobic domain, were found to yield viable virus progeny, but this was without exception accompanied by the emergence of additional mutations within protein C. These point mutations or sequence duplications were located downstream of the engineered deletion and generally increased the hydrophobicity, suggesting that they may compensate for the loss of the central hydrophobic domain. Two of the second-site mutations, together with the corresponding deletion, were introduced into a wild-type genetic backbone, and the analysis of these "double mutants" provided direct evidence that the viability of the deletion mutant indeed depended on the presence of the second-site mutation. Our results corroborate the notion that hydrophobic interactions of protein C are essential for the assembly of infectious flavivirus particles but rule out the possibility that individual residues of the central hydrophobic domain are absolutely required for infectivity. Furthermore, the double mutants were found to be highly attenuated and capable of inducing a protective immune response in mice at even lower inoculation doses than the previously characterized 16-amino-acid-residue deletion mutant, suggesting that the combination of large deletions and second-site mutations may be a superior way to generate safe, attenuated flavivirus vaccine strains.