A computational-experimental approach identifies mutations that enhance surface expression of an oseltamivir-resistant influenza neuraminidase.
A computational-experimental approach identifies mutations that enhance surface expression of an oseltamivir-resistant influenza neuraminidase.
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DOI：
10.1371/journal.pone.0022201
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Baltimore D
中科院分区：
文献类型：
--
作者：
Bloom JD;Nayak JS;Baltimore D
His274Tyr（H274Y）奥司他韦（达菲）耐药突变导致人类季节性H1N1流感早期分离株和猪源性H1N1大流行中表面表达的神经氨酸酶蛋白和活性的总水平大幅下降。在季节性H1N1中，H274Y只是在发生抵消这种减少的继发突变后才变得广泛传播。H274 Y目前在H1N1大流行中是罕见的，并且仍然不清楚是否存在可能类似地抵消与H1N1大流行中这种抗性突变相关的神经氨酸酶表面表达降低的继发突变。在这里，我们调查的可能性预测这样的二次突变。我们首先测试了几种计算方法的能力，以回顾性地识别在奥司他韦耐药性出现前不久，在季节性H1N1中增强表面表达的神经氨酸酶蛋白和活性水平的二次突变。然后，我们使用最成功的计算方法来预测一组候选的大流行性H1N1神经氨酸酶的二级突变。我们通过实验筛选这些突变，发现其中几个确实部分抵消了H274 Y引起的神经氨酸酶表面表达的减少。二次突变中的两个一起将表面表达的神经氨酸酶活性恢复到野生型水平，并且还消除了由H274 Y引起的组织培养中病毒生长的非常轻微的降低。因此，我们的工作证明了一种结合计算实验的方法，用于识别增强神经氨酸酶表面表达的突变，并描述了几个特定的突变，有可能是相关的奥司他韦耐药性在大流行性H1N1的传播。
The His274Tyr (H274Y) oseltamivir (Tamiflu) resistance mutation causes a substantial decrease in the total levels of surface-expressed neuraminidase protein and activity in early isolates of human seasonal H1N1 influenza, and in the swine-origin pandemic H1N1. In seasonal H1N1, H274Y only became widespread after the occurrence of secondary mutations that counteracted this decrease. H274Y is currently rare in pandemic H1N1, and it remains unclear whether secondary mutations exist that might similarly counteract the decreased neuraminidase surface expression associated with this resistance mutation in pandemic H1N1. Here we investigate the possibility of predicting such secondary mutations. We first test the ability of several computational approaches to retrospectively identify the secondary mutations that enhanced levels of surface-expressed neuraminidase protein and activity in seasonal H1N1 shortly before the emergence of oseltamivir resistance. We then use the most successful computational approach to predict a set of candidate secondary mutations to the pandemic H1N1 neuraminidase. We experimentally screen these mutations, and find that several of them do indeed partially counteract the decrease in neuraminidase surface expression caused by H274Y. Two of the secondary mutations together restore surface-expressed neuraminidase activity to wildtype levels, and also eliminate the very slight decrease in viral growth in tissue-culture caused by H274Y. Our work therefore demonstrates a combined computational-experimental approach for identifying mutations that enhance neuraminidase surface expression, and describes several specific mutations with the potential to be of relevance to the spread of oseltamivir resistance in pandemic H1N1.
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