Directed mutagenesis of a Sindbis virus pathogenesis site.
Directed mutagenesis of a Sindbis virus pathogenesis site.
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辛德比斯病毒发病位点的定向诱变。
DOI：
10.1006/viro.1993.1111
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发表时间：
1993
期刊：
影响因子：
3.7
通讯作者：
Johnston,RE
中科院分区：
文献类型：
--
作者：
Schoepp,RJ;Johnston,RE
Sindbis病毒的E2糖蛋白基因包含三个中和抗原位点，对其中一个E2c位点的单克隆抗体（MAb）逃逸突变体的检测表明，E2c结构域与新生小鼠毒力的决定因素有关。与E2c位点相关的密码子E2 62、E2 96和E2 159进行定点诱变，产生25个在这些位点上含有不同氨基酸的活病毒突变体文库。在酶联免疫吸附试验中评估突变体结合E2a、E2b和E2c单克隆抗体的能力，在皮下或颅内接种后诱导新生小鼠的致命疾病，并与野生型病毒竞争结合未分离的新生小鼠脑细胞。这些替换均不影响E2a或E2b单抗的结合，并且与E2c单抗结合减少与新生小鼠毒力降低相关。然而，没有观察到毒力和与E2c单克隆抗体结合的降低与野生型病毒与小鼠脑细胞结合的竞争减少的相关性。突变对E2c结合和毒力的影响很大程度上取决于基因座和特异性替代，这提示了活病毒疫苗的分子设计策略，即选择基因座和密码子以最大限度地衰减和最小化恢复毒力的可能性。
The E2 glycoprotein gene of Sindbis virus contains three neutralizing antigenic sites, and examination of monoclonal antibody (MAb) escape mutants of one of these, the E2c site, has suggested an association of the E2c domain with determinants of virulence in neonatal mice. Codons associated with the E2c site, E2 62, 96, and 159, were subjected to site-directed mutagenesis generating a library of 25 viable virus mutants containing different amino acids at these loci. The mutants were assessed for their ability to bind E2a, E2b, and E2c MAbs in an enzyme-linked immunosorbent assay, to induce fatal disease in neonatal mice following subcutaneous or intracranial inoculation and to compete with wild-type virus for binding to unfractionated neonatal mouse brain cells. None of the substitutions affected binding of E2a or E2b MAbs, and decreased binding to E2c MAbs was correlated with decreased virulence in neonatal mice. However, correlation of decreased virulence and binding to E2c MAbs with decreased competition for wild-type virus binding to mouse brain cells was not observed. The effect of mutation on E2c binding and virulence varied widely depending on the locus and the specific substitution, suggesting strategies for the molecular design of live virus vaccines which select both locus and codon to maximize attenuation and minimize the potential for reversion to virulence.