Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111
Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111
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DOI：
10.3390/v12010105
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发表时间：
2020-01-01
期刊：
影响因子：
4.7
通讯作者：
Sabeti, Pardis C.
中科院分区：
文献类型：
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作者：
Lin, Aaron E.;Diehl, William E.;Sabeti, Pardis C.
对于高致病性病毒，迫切需要能够快速进行的报告分析，以确定潜在的功能突变，以便在最大限度的遏制下(例如，生物安全级别4[BSL-4])进行进一步研究。埃博拉病毒核蛋白(NP)在病毒生命周期中发挥着多种重要作用，但很少有工具研究该蛋白在BSL-2或等效包涵体下的作用。因此，我们采用报告分析法来测量活细胞中NP的寡聚和病毒粒子样颗粒(VLP)的产生，并进一步使用已建立的微型基因组分析来测量转录和复制。作为概念验证，我们检查了NP-R111C替代，它出现在2013-2016年西非埃博拉病毒疾病流行期间，并上升到高频率。NP-R111C使NP寡聚和VLP萌发略有增加，但转录和复制略有减少。相反，一个人工合成的电荷反转突变体NP-R111E大大增加了寡聚化，但抑制了转录和复制。根据NP低聚物的最新结构，这些结果是有趣的，这表明邻近的残基K110与邻近的NP分子上的E349形成盐桥。通过发展和利用多种报告分析方法，我们发现NP-111位置在NP在蛋白质结构、病毒粒子萌发以及转录和复制中的作用之间起着复杂的相互作用。
For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measured transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 2013-2016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NP's roles in protein structure, virion budding, and transcription and replication.