Biochemistry Department and National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI, 53706, USA.
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
Antiviral Res. 2020 Aug;180:104822. doi: 10.1016/j.antiviral.2020.104822. Epub 2020 May 21.
The Ebola Virus is a causative agent of viral hemorrhagic fever outbreaks and a potential global health risk. The outbreak in West Africa (2013-2016) led to 11,000+ deaths and 30,000+ Ebola infected individuals. The current outbreak in the Democratic Republic of Congo (DRC) with 3000+ confirmed cases and 2000+ deaths attributed to Ebola virus infections provides a reminder that innovative countermeasures are still needed. Ebola virus encodes 7 open reading frames (ORFs). Of these, the nucleocapsid protein (eNP) encoded by the first ORF plays many significant roles, including a role in viral RNA synthesis. Here we describe efforts to target the C-terminal domain of eNP (eNP-CTD) that contains highly conserved residues 641-739 as a pan-Ebola antiviral target. Interactions of eNP-CTD with Ebola Viral Protein 30 (eVP30) and Viral Protein 40 (eVP40) have been shown to be crucial for viral RNA synthesis, virion formation, and virion transport. We used nuclear magnetic response (NMR)-based methods to screened the eNP-CTD against a fragment library. Perturbations of 1D H NMR spectra identified of 48 of the 439 compounds screened as potential eNP CTD interactors. Subsequent analysis of these compounds to measure chemical shift perturbations in 2D H,N NMR spectra of N-labeled protein identified six with low millimolar affinities. All six perturbed an area consisting mainly of residues at or near the extreme C-terminus that we named "Site 1" while three other sites were perturbed by other compounds. Our findings here demonstrate the potential utility of eNP as a target, several fragment hits, and provide an experimental pipeline to validate viral-viral interactions as potential panfiloviral inhibitor targets.
埃博拉病毒是病毒性出血热疫情的病原体，也是潜在的全球健康威胁。2013 年至 2016 年西非的疫情导致 11000 多人死亡，30000 多人感染埃博拉病毒。目前刚果民主共和国（DRC）的疫情已导致 3000 多例确诊病例和 2000 多人死亡，这提醒人们仍需要采取创新的对策。埃博拉病毒编码 7 个开放阅读框（ORFs）。其中，第一个 ORF 编码的核衣壳蛋白（eNP）发挥了许多重要作用，包括在病毒 RNA 合成中的作用。在这里，我们描述了针对 eNP 的 C 末端结构域（eNP-CTD）的靶向努力，该结构域包含高度保守的残基 641-739，作为泛埃博拉抗病毒靶标。已证明 eNP-CTD 与埃博拉病毒蛋白 30（eVP30）和病毒蛋白 40（eVP40）的相互作用对于病毒 RNA 合成、病毒粒子形成和病毒粒子运输至关重要。我们使用基于核磁共振（NMR）的方法对 eNP-CTD 进行了筛选。1D H NMR 光谱的扰动鉴定了 439 种筛选化合物中的 48 种可能是 eNP CTD 相互作用者。随后，对这些化合物进行分析，以测量 2D H，N NMR 光谱中 N 标记蛋白的化学位移扰动，鉴定出 6 种具有低毫摩尔亲和力的化合物。这 6 种化合物都干扰了一个主要由极端 C 末端附近或附近的残基组成的区域，我们将其命名为“Site 1”，而其他三个位点则被其他化合物干扰。我们的研究结果证明了 eNP 作为靶标的潜在用途，发现了几个片段命中，并提供了一个实验管道来验证病毒-病毒相互作用作为潜在的泛 filoviral 抑制剂靶标。
