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.

中文翻译：

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对埃博拉病毒核蛋白寡聚化、病毒粒子样颗粒出芽和小基因组活性的报告分析揭示了核蛋白氨基酸 111 位的重要性。


对于高致病性病毒，迫切需要能够快速进行的报告分析，以识别潜在的功能突变，以便在最大程度的遏制（例如生物安全级别 4 [BSL-4]）下进行进一步研究。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，但在 BSL-2 或同等遏制条件下研究该蛋白质的工具却很少。因此，我们采用报告基因测定法来测量活细胞中 NP 寡聚化和病毒颗粒样颗粒 (VLP) 的产生，并使用已建立的小基因组测定法进一步测量转录和复制。作为概念验证，我们检查了 NP-R111C 替代，该替代在 2013 年至 2016 年西非埃博拉病毒疾病流行期间出现并上升到高频率。 NP-R111C 略微增加 NP 寡聚化和 VLP 出芽，但略微降低转录和复制。相比之下，合成的电荷反转突变体 NP-R111E 大大增加了寡聚化，但消除了转录和复制。鉴于 NP 寡聚物的最新结构，这些结果很有趣，该结构揭示了相邻残基 K110 与相邻 NP 分子上的 E349 形成盐桥。通过开发和利用多种报告基因检测，我们发现 NP-111 位置介导 NP 在蛋白质结构、病毒体出芽以及转录和复制中的作用之间的复杂相互作用。