Mutational analysis of a neutralization epitope on the dengue type 2 virus (DEN2) envelope protein: Monoclonal antibody resistant DEN2/DEN4 chimeras exhibit reduced mouse neurovirulence
Mutational analysis of a neutralization epitope on the dengue type 2 virus (DEN2) envelope protein: Monoclonal antibody resistant DEN2/DEN4 chimeras exhibit reduced mouse neurovirulence
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DOI：
10.1006/viro.1996.0550
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发表时间：
1996-10-15
期刊：
影响因子：
3.7
通讯作者：
Lai, CJ
中科院分区：
文献类型：
--
作者：
Hiramatsu, K;Tadano, M;Lai, CJ
通过突变分析研究了登革热 2 型病毒 (DEN2) 中和单克隆抗体 (mab) 3H5 的抗原位点。序列比较表明，以前被认为代表 DEN2 特异性 mab 3H5 结合位点的 DEN2 包膜糖蛋白 (E) 从位置 386 延伸到 397 的大部分 12 氨基酸序列也存在于一些登革热 1、3 或 4 型病毒株中。然而，上游第383至385位Glu-Pro-Gly序列占据的区域在DEN2毒株中完全保守，但在其他血清型病毒中有所不同，表明该序列可能是malc 3H5抗原位点的一部分。我们通过使用先前构建的嵌合 DEN2(PreM-E)/DEN4 cDNA 克隆来研究这种可能性，以产生带有 DEN2 PreM 和 E 序列的可行突变体，可以分析这些突变体与 mab 3H5 的结合和中和。我们构建了 13 个这样的 DEN2 突变体，其在 DEN2 E 位置 383 和 393 之间的区域中含有单个氨基酸取代。跨越 DEN2 位置 386 到 393 的区域中的每个单个取代产生了与亲代嵌合病毒一样与 mab 3H5 反应的病毒。这些结果与该区域的序列保守程度一致。相反，如通过免疫荧光测定检测到的，在位置383、384或385处持续氨基酸取代的6个突变体中有5个未能与mab 3H5反应并且未能被mab中和。有趣的是，当大脑内接种时，与亲本嵌合 DEN2 相比，5 种 mab 抗性 DEN2 突变体中的每一种都表现出小鼠神经毒力降低。这些观察结果表明，DEN2 E 位置 383-385 处的 Glu-Pro-Gly 序列是 mab 3H5 所针对的位点的组成部分。在最近确定的相关蜱传脑炎病毒E的三维结构中，Glu-Pro-Gly序列将位于拟与宿主细胞受体结合的免疫球蛋白样结构域的侧面。 (C) 1996 学术出版社
The antigenic site of dengue type 2 virus (DEN2)-neutralizing monoclonal antibody (mab) 3H5 was investigated by mutational analysis. Sequence comparisons indicated that much of the 12-amino-acid sequence extending from position 386 to 397 of the DEN2 envelope glycoprotein (E) previously thought to represent the DEN2-specific mab 3H5 binding site was also present in some dengue type 1, 3, or 4 virus strains. However, the region occupied by the Glu-Pro-Gly sequence at upstream positions 383 to 385 was completely conserved among DEN2 strains, but divergent in other serotype viruses, suggesting that this sequence might be part of the antigenic site of malc 3H5. We investigated this possibility by employing the previously constructed chimeric DEN2(PreM-E)/DEN4 cDNA clone to produce viable mutants bearing DEN2 PreM and E sequences that could be analyzed for binding to and neutralization by mab 3H5. We constructed 13 such DEN2 mutants that contained a single amino acid substitution in the region between positions 383 and 393 of DEN2 E. Each single substitution in the region spanning positions 386 through 393 of DEN2 yielded a virus that was as reactive with mab 3H5 as the parental chimeric virus. These results are consistent with the extent of sequence conservation in the region. In contrast, 5 of 6 mutants that sustained an amino acid substitution at position 383, 384, or 385 failed to react with mab 3H5 as detected by immunofluorescence assay and failed to be neutralized by the mab. Interestingly, each of the 5 mab-resistant DEN2 mutants also exhibited reduced mouse neurovirulence compared to parental chimeric DEN2 when inoculated intracerebrally. These observations suggest that the Glu-Pro-Gly sequence at positions 383-385 of the DEN2 E is a component of the site against which mab 3H5 is directed. In the recently determined three-dimensional structure of the related tick-borne encephalitis virus E, the Glu-Pro-Gly sequence would be located on the lateral surface of the immunoglobulin-likedomain that is proposed to bind to the host cell receptor. (C) 1996 Academic Press, Inc.