NSF/MCB-BSF: Revealing the steps and modulators of coronavirus fusion using single-molecule tools

许多病毒的感染始于病毒被膜和宿主细胞膜之间的膜融合事件，导致病毒基因组转移到宿主细胞中。通常，这一过程是由病毒融合蛋白介导的。在这个项目中，我们检测了冠状病毒刺突蛋白。在SPEK中，当融合肽插入宿主膜中时，它启动膜融合。为了研究这一过程，将开发单分子工具和技术，并用于研究融合肽与模仿不同种类宿主细胞的膜表面之间的相互作用。我们的目标是利用互补技术了解融合肽序列和靶膜之间的关系，从而能够跨尺度地检查这一过程，从融合肽到整个病毒。随着对调节这种关键相互作用的化学特征背后的科学有了更好的理解，可以做出新的病毒适应新宿主的预测，并利用这些预测来阻止这一过程。该项目的更广泛影响包括该工作的内在价值，因为将获得有用的信息，可用于设计新型抗病毒药物，并确定为宿主对病毒进入的易感性预测提供信息的化学规则。鉴于主要的融合成员在冠状病毒家族中高度保守，这些研究将直接适用于所有冠状病毒，包括那些尚未出现的冠状病毒。此外，还将为研究生提供培训机会，并为高中生提供外联活动。对于冠状病毒来说，进入宿主细胞是由从其膜被膜突出的单一糖蛋白介导的，称为刺突(S)。决定冠状病毒传播能力的一个关键因素是S与其靶宿主膜的相互作用。在S体内，直接与膜相互作用的区域称为融合肽，Fp。正是FP与宿主膜的物理化学作用将其锚定，从而使膜发生必要的变形，导致膜融合并将病毒基因组运送到细胞内。因此，在纳米尺度上了解FP与宿主细胞的化学偶联将有助于制定限制这些相互作用以阻止感染的策略，同时也能够预测新出现的菌株和敏感宿主的特征。本项目的目标是确定和测量负责将FP插入膜的特定分子间相互作用。单分子工具和技术将被开发、扩展和使用，以了解宿主膜和FP之间的基本化学密码，这些密码调节控制融合过程的相互作用。这一项目的智力价值在于发现了宿主膜化学与驱动病毒与宿主之间分子尺度相互作用的FP氨基酸序列之间的关系。该项目由分子和细胞生物科学部的细胞动力学和功能以及分子生物物理学计划共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Infection by many viruses commences with a membrane fusion event between the viral envelope and host cell membrane, which leads to the transfer of the viral genome into the host cell. Typically, this process is mediated by viral fusion proteins. In this project, the coronavirus spike protein is examined. Within spike, the fusion peptide initiates membrane fusion when it inserts into the host membrane. To study this process, single molecular tools and techniques will be developed and used to study the interaction between the fusion peptide with membrane surfaces that mimic different kinds of host cells. The goal is to understand the relationship between fusion peptide sequence and target membranes using complementary techniques that enable examination of this process across scales, from the fusion peptide to the whole virus. With a better understanding of the science behind the chemical features that modulate this critical interaction, new predictions for virus adaptation to new hosts can be made and exploited to block the process. The Broader Impacts of this project include the intrinsic merit of the work as useful information will be gained that can be leveraged for the design of novel antiviral drugs, and to identify chemical rules that inform predictions of host susceptibility to viral entry. Given that major fusion players are highly conserved across the CoV family, these studies will be directly applicable to all CoVs, including those yet to emerge. In addition, training opportunities for graduate students will be provided, along with outreach activities for high school students. For coronavirus, entry into a host cell is mediated by a single glycoprotein protruding from its membrane envelope, called spike (S). A key determinant of the ability of coronavirus to spread is the interaction of S with its target host membrane. Within S, the region that directly interacts with the membrane is called the fusion peptide, FP. It is the physico-chemical interactions of the FP with the host membrane that anchors it, consequently enabling the necessary deformations of the membrane that leads to membrane fusion and the delivery of the viral genome into the cell. Thus, understanding chemical coupling of the FP with the host cell at the nanoscale will facilitate the development of strategies to limit those interactions to stop infection, but also to enable predicting the characteristics of emerging strains and the susceptible hosts. The objective of this project is to identify and measure the specific intermolecular interactions responsible for insertion of FP into membranes. Single molecule tools and techniques will be developed, expanded, and used to understand the fundamental chemical code between the host membrane and FP that modulate interactions that control the fusion process. The intellectual merit of this project is discovering the relationship between the host membrane chemistry and amino acid sequence of the FP that drive the molecular scale interactions between virus and host. This project is co-funded by the Cellular Dynamics and Function together with the Molecular Biophyics programs, both in the Division for Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.