Biology of ebolavirus inclusion bodies

埃博拉病毒是引起出血热的重要人类病原体，病死率高。不幸的是，虽然我们最近看到在开发治疗方法方面取得了巨大进展，但即使在接受治疗的患者中，病例死亡率仍然很高，这强调了在这一领域取得进一步进展的必要性。因此，我们有兴趣更好地了解这些病毒的生命周期，以确定新的抗病毒药物的靶标，特别是那些针对病毒-宿主界面的抗病毒药物。在感染期间，埃博拉病毒蛋白质积累形成称为包涵体（IBs）的结构，我们和其他人之前已经证明，包涵体作为病毒基因组复制和转录的位点，以及核衣壳的组装，在病毒生命周期中发挥重要作用，核衣壳将病毒基因组藏在病毒颗粒内。对于许多其他非节段负义RNA病毒（nnsv），类似的IBs现在已被认为是液体细胞器，即不通过描绘膜来定义的微环境，而是通过液-液相分离将其结合在一起。事实上，基于对感染埃博拉病毒的细胞的活细胞成像分析，我们之前发现埃博拉病毒IBs也显示出液体细胞器样特征。然而，我们目前对埃博拉病毒IB的特性、控制其形成的因素、液体细胞器样特性如何促进IB功能，或特定病毒和/或细胞因子在这些过程中发挥的作用知之甚少。为了更好地了解这些重要结构的形成和功能，我们首先计划根据包涵体成分的扩散系数和IBs对渗透休克的易感性等物理特征，研究埃博拉病毒IBs是否确实是液体细胞器。然后，我们将确定哪些病毒蛋白质-蛋白质和/或蛋白质- rna相互作用有助于它们的形成，并评估相关区域的生化特性的贡献。此外，我们想要了解核衣壳是如何从包涵体中输出的，特别是由于核衣壳凝聚引起的相分离是否在这一过程中起作用。最后，由于EBOV在其生命周期的许多方面都篡夺了宿主蛋白，因此我们希望使用基于质谱的方法组合来表征与EBOV IBs相关的宿主蛋白，并选择已确定的因素直接评估它们在IBs的形成和功能中所起的作用。这些研究将增加我们对IB生物学的了解，这是病毒学中的一个新兴课题，不仅针对埃博拉病毒，而且针对整个nnsv，并将提供有关针对这些病毒生命周期重要方面的抗病毒药物可能的分子靶点的信息。

Ebolaviruses are important human pathogens causing hemorrhagic fevers with high case fatality rates. Unfortunately, while we have recently seen a tremendous progress in the development of treatments, case fatality rates remains high even in treated patients, emphasizing the need for further progress in this field. Therefore, we are interested in better understanding the life cycle of these viruses in order to define targets for novel antivirals, particularly those aimed at the virus-host interface.During infection, ebolavirus proteins accumulate to form structures called inclusion bodies (IBs), which we and others have previously shown to play an essential function in the virus life cycle as sites of viral genome replication and transcription, as well as the assembly of nucleocapsids, which harbor the viral genome inside virus particles. For a number of other non-segmented negative-sense RNA viruses (NNSVs) similar IBs have now been recognized as being liquid organelles, i.e. microenvironments that are not defined by delineating membranes, but are rather held together by liquid-liquid phase separation. Indeed, based on live cell imaging analysis of cells infected with ebolaviruses we have previously found that ebolavirus IBs also display features that suggest a liquid organelle-like character. However, we currently know little about the properties of ebolavirus IBs, what governs their formation, how liquid organelle-like properties may contribute to IB function, or what role specific viral and/or cellular factors play in these processes.In order to better understand both the formation and function of these important structures, we first plan to investigate whether ebolavirus IBs are indeed liquid organelles, based on physical characteristics such as the diffusion coefficient of inclusion body constituents, and susceptibility of IBs to osmotic shock. We will then determine which viral protein-protein and/or protein-RNA interactions contribute to their formation and assess the contributions of the biochemical characteristics of the involved regions. Further, we want to understand how nucleocapsids are exported from inclusion bodies, and particularly whether phase separation due to nucleocapsid condensation plays a role in this process. Finally, since EBOV usurps host proteins for many aspects of its life cycle, we want to characterize the host proteins associated with EBOV IBs using a combination of mass spectrometry-based approaches, and for a selection of the identified factors directly assess the role they play in both the formation and function of IBs. These studies will increase our knowledge of IB biology, which is an emerging topic in virology, not only for ebolaviruses, but also for NNSVs as a whole, and will provide information regarding possible molecular targets for antivirals targeting an essential aspect of the life cycle of these viruses.