Structural and functional analysis of the coronavirus spike protein fusion peptide

项目摘要/摘要 被包裹的病毒通过与质膜上的受体结合，然后经历 与宿主膜融合。结合和融合都是由一种特定的病毒“尖峰”蛋白介导的，即 通常通过蛋白水解性切割为融合激活做好准备，以暴露融合多肽。冠状病毒融合 SPEKE蛋白(冠状病毒S)是一种复杂的生物分子机器，具有新颖的融合肽和大量的 在其聚变反应中具有固有的灵活性。这些病毒在不同的入侵途径中利用了这一点 是病毒嗜性的主要决定因素。我们率先提出了这样一个概念，即蛋白质分解的切割事件 在S中，导致膜融合发生在受体结合(S1)和融合(S2)结构域的界面上 (称为S1/S2)，以及与S2内结构和功能上新的融合肽(称为S2‘)相邻。 因此，冠状病毒S与大多数其他I类融合蛋白有显著的差异，包括：1) 释放融合肽的蛋白分解事件是多样的，以及2)融合肽本身在 与其他融合肽的序列比较，含有重要的疏水性和负性- 带电残基，可能代表比正常大的融合“平台”，而不是定义的“肽”。因此， 融合肽的活性可能受融合平台的重组控制，基于两者的疏水性 (即脂质结合)和离子(即钙离子)相互作用。尽管最近出现了S结构在它们的前 在融合状态下，对冠状病毒家族的膜融合机制的了解仍然非常有限，或者 任何结构信息都可以将S的结构生物学方面与其在膜融合中的功能联系起来。这 信息对于了解病毒的发病机制和冠状病毒在人群中的出现至关重要。我们 提出了一种综合的生物物理、生化和活体方法来研究独特的裂解激活 中东呼吸综合征冠状病毒与重症急性冠状病毒对S蛋白的调控 以呼吸综合征冠状病毒(SARS-CoV)为主要模型。我们将使用最先进的光谱学 以及一种创新的单粒子示踪技术来研究S蛋白融合肽的功能，并将这些 包括BSL3在内的体内传染性研究将使人们能够完整地了解冠状病毒融合激活的情况。这些 方法将揭示结构和功能如何根据S的关键激活因子而变化；即受体结合， 酶的可获得性和当地离子环境。这些研究将使我们能够确定共同的原则 这可以应用于所有CoV，通过这些创新研究推动该领域的发展将提供关键的 关于保护人类健康所需的冠状病毒进入和嗜性的知识可能是一种新的病原体 导致严重暴发，而对此几乎没有或根本没有医学对策。

Project Summary / Abstract Enveloped viruses access their host cells by binding to receptors on the plasma membrane and then undergoing fusion with the host membrane. Both binding and fusion are mediated by a specific viral “spike” protein that is typically primed for fusion activation by proteolytic cleavage to expose the fusion peptide. Coronavirus fusion spike protein (CoV S) is a complex biomolecular machine that has a novel fusion peptide with has a great deal of inherent flexibility in its fusion reaction. This is exploited by these viruses in their diverse entry pathways and is a primary determinant of viral tropism. We have pioneered the concept that that the proteolytic cleavage events in S that lead to membrane fusion occur both at the interface of the receptor binding (S1) and fusion (S2) domains (called S1/S2), as well as adjacent to a structurally and functionally novel fusion peptide within S2 (called S2’). Thus, there are notable differences between CoV S and most other class I fusion proteins including: 1) that the proteolytic events liberating the fusion peptide are diverse, and 2) that the fusion peptide itself is atypical in sequence compared to other fusion peptides, containing a mixture of important hydrophobic and negatively- charged residues, and may represent a larger than normal fusion “platform” instead of a defined “peptide”. Thus fusion peptide activity is likely controlled by reorganization of the fusion platform, based on both hydrophobic (i.e. lipid-binding) and ionic (i.e. Ca2+) interactions. Despite the recent availability of S structures in their pre- fusion state, there remains a very limited mechanistic understanding of membrane fusion for the CoV family, or any structural information to correlate structural biology aspects of S to its function in membrane fusion. This information is critical to understanding viral pathogenesis and CoV emergence into the human population. We propose an integrated biophysical, biochemical, and in vivo approach to study the unique cleavage-activated regulation of CoV S protein, using Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) as primary models. We will use state-of-the-art spectroscopy and an innovative single particle tracking technique to study S protein fusion peptide function, and combine these with in vivo infectivity studies, including at BSL3, will allow a complete picture of CoV fusion activation. These approaches will reveal how structure and function vary depending on the key activators of S; i.e. receptor binding, protease availability and the local ionic environment. These studies will allow us to determine common principals that can be applied to all CoVs, moving the field forward with these innovative studies will provide critical knowledge about CoV entry and tropism needed to safeguard human health from an emerging pathogen likely to cause severe outbreaks, and for which few or no medical countermeasures exist.