Structural and functional analysis of the coronavirus spike protein fusion peptide

项目总结/摘要 有包膜病毒通过与质膜上的受体结合，然后经历 与宿主细胞膜融合。结合和融合都是由一种特定的病毒“刺突”蛋白介导的， 通常通过蛋白水解切割暴露融合肽来引发融合活化。冠状病毒融合 刺突蛋白（CoVS）是一种复杂的生物分子机器，具有一种新型的融合肽， 聚变反应中固有的灵活性这些病毒在其不同的进入途径中利用了这一点， 是病毒嗜性的主要决定因素。我们开创了蛋白水解裂解事件 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 （称为S1/S2），以及邻近S2内的结构和功能新颖的融合肽（称为S2 '）。 因此，在CoV S和大多数其他I类融合蛋白之间存在显著差异，包括：1）CoV S与大多数I类融合蛋白相比，具有更高的生物学活性。 释放融合肽的蛋白水解事件是多样的，和2）融合肽本身在免疫学中是非典型的。 序列相比，其他融合肽，含有重要的疏水性和负- 带电荷的残基，并且可以代表比正常融合“平台”更大的融合“平台”而不是限定的“肽”。因此 融合肽的活性很可能是通过融合平台的重组来控制的，这是基于疏水性和非疏水性两个方面。 (i.e.脂质结合）和离子（即Ca 2+）相互作用。尽管最近S结构在其预- 融合状态，对CoV家族的膜融合机制的理解仍然非常有限，或者 将S的结构生物学方面与其在膜融合中的功能相关联的任何结构信息。这 这些信息对于理解病毒发病机制和CoV进入人群至关重要。我们 提出了一个综合的生物物理，生物化学和体内的方法来研究独特的裂解激活 使用中东呼吸综合征冠状病毒（MERS-CoV）和严重急性 呼吸综合征冠状病毒（SARS-CoV）作为主要模型。我们将使用最先进的光谱学 和创新的单粒子跟踪技术来研究S蛋白融合肽功能，并将这些联合收割机 通过体内感染性研究，包括在BSL 3，将允许CoV融合激活的完整图片。这些 这些方法将揭示结构和功能如何根据S的关键活化剂而变化;即受体结合， 蛋白酶可用性和局部离子环境。这些研究将使我们能够确定共同的原则 可以应用于所有CoV，通过这些创新研究推动该领域向前发展，将提供关键的 了解冠状病毒的进入和嗜性，以保护人类健康免受新出现的病原体的侵害， 导致严重的疾病爆发，并且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.