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Characterization of Stabilizing Mutations in the H5N1 Hemagglutinin Influenza Protein.

机译：H5N1血凝素流感蛋白中稳定突变的特征。

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摘要

While the number of human infections with highly pathogenic avian H5N1 influenza viruses continues to rise, these viruses are still unable to transmit efficiently between humans. Recently, we and others introduced mutations into the receptor-binding domain of the influenza viral surface glycoprotein hemagglutinin (HA) that altered the receptor-binding preference of HA from avian- to human-type, but also reduced HA protein stability. Virus passages in ferrets resulted in the emergence of compensatory mutations that reduced the pH of fusion and stabilized HA. The H5 viruses that possessed human-type receptor-binding specificity and additional stabilizing mutations in HA could transmit among ferrets through respiratory droplets. To identify additional stabilizing mutations in H5 HAs, we constructed and screened a mutant virus library possessing two mutations that confer binding to human-type receptors in addition to random mutations in the HA ectodomain. After several rounds of heat treatment and virus amplification, we isolated several mutants with increased thermostability. These mutants also initiated membrane fusion at a lower pH value that was comparable to that of human HA proteins and maintained their human-type receptor-binding property.;In addition, we analyzed publicly available H5 HA sequences for polymorphisms in H5N1 human isolates that were not present in avian isolates. This analysis revealed 59 unique polymorphisms. To date, we have tested a subset of these candidates and identified one mutation that affects HA stability. Our previous ferret transmission study was carried out with an HA protein of clade 1. Most currently circulating H5N1 viruses belong to clade 2 subclades. Therefore, we introduced the transmission-conferring mutations into eight genetically distinct clade 2 viruses. All mutant viruses bound to human-type receptors and underwent membrane fusion at the same pH or a more acidic pH than the corresponding wild-type HA. Overall, these variants match the phenotype of the H5 virus that transmitted via respiratory droplets among ferrets. Collectively, the presented data expand on the understanding of HA stability which has been recently identified to play a key role in the mammalian respiratory droplet transmission of influenza viruses.
机译：尽管人类感染高致病性禽H5N1流感病毒的数量继续增加，但这些病毒仍无法在人与人之间有效传播。最近，我们和其他人将突变引入了流感病毒表面糖蛋白血凝素（HA）的受体结合域，该突变将HA的受体结合偏好从禽型转变为人型，但同时降低了HA蛋白的稳定性。雪貂在雪貂中的传代导致代偿性突变的出现，代偿性突变降低了融合的pH值并稳定了HA。具有人型受体结合特异性和HA中其他稳定突变的H5病毒可通过呼吸道飞沫在雪貂之间传播。为了鉴定H5 HA中的其他稳定突变，我们构建并筛选了一个突变病毒文库，该文库除了HA胞外域中的随机突变外，还具有两个赋予人型受体结合的突变。经过几轮热处理和病毒扩增后，我们分离出了几个热稳定性增强的突变体。这些突变体还在与人HA蛋白相当的较低pH值下启动了膜融合，并维持了其人型受体结合特性。;此外，我们分析了公开可获得的H5 HA序列中H5N1人类分离株中的多态性。在禽分离物中不存在。该分析揭示了59种独特的多态性。迄今为止，我们已经测试了这些候选基因的一部分，并确定了一种会影响HA稳定性的突变。我们先前的雪貂传播研究是使用进化枝1的HA蛋白进行的。当前传播的大多数H5N1病毒属于进化枝2的子进化枝。因此，我们将赋予传播的突变引入了8种遗传上不同的进化枝2病毒。所有突变型病毒都与人型受体结合，并在与相应的野生型HA相同的pH或更酸性的pH下进行膜融合。总体而言，这些变体与在雪貂之间通过呼吸道飞沫传播的H5病毒的表型匹配。总体而言，所提供的数据扩展了对HA稳定性的理解，而HA稳定性最近已被确定在哺乳动物流感病毒的呼吸道小滴传播中起关键作用。

著录项

  • 作者

    Hanson, Anthony P.;

  • 作者单位

    The University of Wisconsin - Madison.;

  • 授予单位 The University of Wisconsin - Madison.;
  • 学科 Virology.;Epidemiology.;Molecular biology.;Biochemistry.
  • 学位 Ph.D.
  • 年度 2016
  • 页码 120 p.
  • 总页数 120
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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