Tetherin antagonism by the Ebola virus glycoprotein: Molecular mechanism and contribution to viral spread

埃博拉病毒糖蛋白的 Tetherin 拮抗作用：分子机制及其对病毒传播的贡献

• 批准号: 243129136
• 负责人: Professor Dr. Stefan Pöhlmann
• 金额: --
• 依托单位: Institut für Medizinische Virologie und Epidemiologie der Viruskrankheiten
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/243129136?language=en
• 关键词: Tetherin; antagonism; Ebola; virus; glycoprotein

The interferon (IFN) system is an important component of innate immunity and plays a key role in the defence against viral infections. Invading viruses are detected by cellular sensors which trigger the production and secretion of IFN. Binding of IFN to uninfected cells triggers the expression of IFN-induced genes, some of which exert a direct antiviral activity. Tetherin is an IFN-induced protein, which can inhibit release of several enveloped viruses from infected cells. However, some viruses encode tetherin antagonists, which allow viral spread in tetherin expressing cells. The HIV-1 protein Vpu reduces tetherin expression at the cell surface, the site of viral budding. The glycoprotein of Ebola virus (EBOV-GP1,2) also inhibits tetherin, but the underlying mechanism is unclear and will be defined within the proposed studies.Our preliminary data suggest that EBOV-GP1,2, in particular GP2, could relocalize the tetherin N-terminus from the cytoplasm to the extracellular space, and this hypothesis will be investigated. For this, we will clarify if the tetherin N-terminus becomes accessible to antibody binding upon coexpression of EBOV-GP1,2, which would demonstrate that the N-terminus has indeed been relocated into the extracellular space. Control experiments conducted in parallel will ensure that antibody binding is not due to interference of EBOV-GP1,2 with membrane integrity. An alternative hypothesis regarding tetherin antagonism is that EBOV-GP1,2 might prevent tetherin localization in compartments of the cytoplasmic membrane, which are used by EBOV for budding. This scenario will be investigated with the help of superresolution microscopy (STORM) and immunogold labeling and electronmicroscopy. Moreover, mutagenic analysis will be employed to identify amino acid substitutions, which selectively block tetherin antagonism but do not compromise EBOV-GP1,2 expression and EBOV-GP1,2-driven virus cell fusion. Finally, reverse genetics will be employed to mutate GP1,2 in the context of authentic EBOV, in order to address if tetherin antagonism is required for viral spread and pathogenesis. These studies will reveal important insights into viral defense strategies against antiviral host cell proteins and might define novel targets for antiviral intervention.

干扰素系统是天然免疫的重要组成部分，在抵御病毒感染中起着关键作用。入侵的病毒是由细胞传感器检测到的，这些传感器触发干扰素的产生和分泌。干扰素与未感染细胞的结合触发了干扰素诱导的基因的表达，其中一些基因具有直接的抗病毒活性。Tetherin是一种干扰素诱导的蛋白质，可以抑制几种被包裹的病毒从感染细胞中释放。然而，一些病毒编码Tetherin拮抗剂，这允许病毒在Tetherin表达细胞中传播。HIV-1蛋白VPU减少了细胞表面的Tetherin表达，这是病毒萌发的地方。埃博拉病毒的糖蛋白(EBOV-GP1，2)也抑制Tetherin，但其机制尚不清楚，将在拟议的研究中定义。我们的初步数据表明，EBOV-GP1，2，特别是GP2，可以将Tetherin的N-末端从细胞质重新定位到细胞外空间，这一假说将被研究。为此，我们将澄清当EBOV-GP1，2共表达时，Tetherin N-末端是否可被抗体结合，这将证明N-末端确实已重新定位到细胞外空间。平行进行的对照实验将确保抗体结合不是由于EBOV-GP1，2对膜完整性的干扰。关于Tetherin拮抗作用的另一种假说是，EBOV-GP1，2可能阻止Tetherin定位于EBOV用来发芽的细胞质膜区域。我们将借助超分辨显微镜(STORM)、免疫金标记法和电子显微镜对这种情况进行研究。此外，突变分析将用于鉴定氨基酸替代，这些氨基酸替代选择性地阻断Tetherin拮抗作用，但不影响EBOV-GP1，2的表达和EBOV-GP1，2驱动的病毒细胞融合。最后，将利用反向遗传学在正宗EBOV的背景下突变GP1，2，以解决是否需要Tetherin拮抗来实现病毒的传播和致病。这些研究将揭示针对抗病毒宿主细胞蛋白的病毒防御策略的重要见解，并可能确定抗病毒干预的新靶点。