TIM Protein-Mediated Ebola Virus-Host Cell Adhesion: Experiments and Models

病毒引起的疾病范围从普通感冒到致命的高传染性埃博拉病毒。它们的“作案手法”是进入健康细胞，通常就像特洛伊木马一样，通过劫持正常的生理过程，欺骗细胞让它们进入。因此，设计针对病毒的治疗方法的一个主要困难在于，试图阻止病毒进入细胞也可能影响正常的生理过程。例如，埃博拉病毒通过将自己伪装成死亡细胞的碎片（废物或遗骸）来感染健康细胞。正常功能是清除这些碎片的健康细胞错误地感染了埃博拉病毒，从而被感染。但病毒和碎片颗粒之间总是存在一些差异，这表明，如果仔细研究，有可能设计出既能阻止特定病毒进入，又能保持正常生理过程基本完整的治疗方法。为了在这一尝试中取得成功，有必要在实验和理论上定量详细地了解病毒摄取过程，特别是后者，这是本项目的主要目标。在使用显微镜技术测量分子间结合力的独立研究的基础上，该项目的重点是发展对粘附分子（细胞表面上导致细胞与其他细胞或颗粒结合的蛋白质）集合行为的理解，并最终开发一个预测模型，用于整个病毒颗粒在被摄取之前如何附着在细胞上。这项工作的一个重要部分是模拟病毒和细胞颗粒的变形，以便定量测量它们的性质。如果成功，该项目将有助于在病毒进入细胞的生物学模型与病毒和细胞的特性之间建立实验验证的定量联系。该研究项目的跨学科性质将为研究生和本科生提供极好的教育和研究机会。研究人员将与阿伦敦的达芬奇科学中心合作，设计一个新的展览，展示病毒摄取的物理和机械细节，以及这项研究如何可能导致潜在的治疗或治愈方法。（达·芬奇科学中心是一个独立的非营利组织，通过探究促进动手科学学习，强调每个年轻人都可以在科学领域获得充满活力和重要的职业机会，并鼓励所有人保持好奇心和创造力。）该项目的目标是建立一个实验知情的预测和定量模型埃博拉病毒(EBOV)-宿主细胞相互作用的分子通过单病毒水平。虽然EBOV-宿主细胞附着已被证明严重依赖于细胞表面受体与病毒外壳之间相互作用的分子生物物理学，但对于指导开发防止EBOV附着并因此进入细胞的治疗方法至关重要的定量理解是完全缺乏的。最近的工作已经确定了TIM家族蛋白的重要性及其粘蛋白样茎结构域（MLD）的几何和力学性质。在这些最新发现的基础上，利用实验和理论分子生物物理学可以进一步取得进展，揭示EBOV附着于宿主细胞的分子、细胞和生物物理机制的定量理解。在利用单分子力谱实验表征TIM家族蛋白如何与EBOV相互作用的独立研究的基础上，该项目将开发生物物理模型，展示单分子生物力学特性，以及MLD的特性（如长度、刚性和电荷密度）如何控制TIM介导的细胞/病毒膜粘附和吞噬。该模型将分三个阶段开发。1)在分子间尺度上，利用粗粒度布朗动力学模型预测单个TIM-1与病毒膜之间的相互作用势。2)在天平中间,glycocalyx将被添加到细胞表面糖蛋白将被添加在病毒表面建立的角色MLD杆的力学性能,使用过程粒度模型解决了在300 k与布朗动力学。3)在整个病毒的力学和内化,发现在分子间和中间尺度将合并病毒颗粒的规模和可变形的膜会增加,目的是利用半解析模型和粗粒度模型的结合，描述病毒的粘附过程，包括膜弯曲和张力的影响。因此，该项目将首次定量阐明ebov -宿主细胞相互作用的生物物理机制，为抗病毒药物开发提供潜在的新靶点。虽然本项目的重点是EBOV，但所采取的方法将适用于其他相关的病毒-宿主细胞相互作用。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Viruses cause diseases ranging from the common cold to the deadly and highly infectious Ebola disease. Their "modus operandi" is to enter healthy cells, often like a Trojan Horse, by hijacking normal physiological processes, tricking the cell to let them in. For this reason, a principal difficulty in designing therapies against viruses lies in the fact that attempts to stop them from entering a cell are also likely to affect normal physiological processes. For example, the Ebola virus infects healthy cells by disguising itself as debris (wastes or remains) from dead cells. Healthy cells whose normal function is to clear up this debris mistakenly take up Ebola and are thus infected. But there are always some differences between a virus and debris particles suggesting that, if studied carefully, it might be possible to design therapies that can block specific virus entry while leaving normal physiological processes essentially intact. In order to be successful in this attempt, it is necessary to understand virus uptake processes in quantitative detail both experimentally and theoretically, and particularly the latter, which is the main goal of this project. Building on separate studies that use microscope based technologies to measure binding forces between molecules, the focus of this project is to develop understanding of the behavior of collections of adhesion molecules (proteins on cell surfaces that cause cells to bind to other cells or particles), and ultimately to develop a predictive model for how an entire virus particle attaches to a cell prior to its uptake. An important part of this work is modeling the deformation of virus and cell particles in order to quantitatively measure their properties. If successful, this project will contribute to establishing an experimentally validated, quantitative connection between biology based models for virus entry into cells and the properties of the virus and the cell. The interdisciplinary nature of this research program will provide an excellent educational and research opportunity for graduate and undergraduate students. Working with the Da Vinci Science Center in Allentown, the investigators will design a new exhibit demonstrating the physical and mechanical details of virus uptake and how its study could lead to potential therapies or a cure. (The Da Vinci Science Center is an independent non-profit organization that promotes hands-on science learning through inquiry, highlights vibrant and important career opportunities in science available to every young person, and encourages all people to be curious and creative.) The goal of this project is to establish an experimentally informed predictive and quantitative model of the Ebola Virus (EBOV)-host cell interactions at the molecular through single-virus levels. While EBOV-host cell attachment has been shown to depend critically on the molecular biophysics of interaction between receptors on the cell surface and the outer coat of the virus, the quantitative understanding essential for guiding the development of therapies that would prevent EBOV from attaching to, and thus from entering a cell, is completely lacking. Recent work has established the importance of TIM family proteins and the geometry and mechanical properties of its mucin-like stalk domain (MLD). Building on these recent findings, further progress can be made by using experimental and theoretical molecular biophysics to uncover a quantitative understanding of the molecular, cellular, and biophysical mechanisms of EBOV attachment to a host cell. Building on separate studies that utilize single-molecule force spectroscopy to characterize experimentally how TIM family proteins interact with EBOV, this project will develop biophysical models that show how single-molecule biomechanical properties, and how the properties of the MLD, such as its length, rigidity, and charge density, control TIM mediated cellular/viral membrane adhesion and engulfment. The model will be developed in three phases. 1) At the Intermolecular Scale, the adhesion between a single TIM-1 and the viral membrane is studied using coarse-grained Brownian Dynamics Models to predict interaction potentials. 2) At the Intermediate Scale, the glycocalyx will be added to the cell surface and glycoproteins will be added on the virus surface to establish the role of the mechanical properties of the MLD stalk, using a course grained model solved with Brownian Dynamics at 300K. 3) At the Mechanics of Whole Virus and Internalization Scale, findings at the Intermolecular and Intermediate Scales will be incorporated at the scale of the viral particles and deformable membranes will be added, with the goal of describing the virus adhesion process, including effects due to membrane bending and tension, using a combination of semi-analytic models and coarse-grained models. The project will thus elucidate quantitatively - for the first time - the biophysical mechanism of EBOV-host cell interaction, providing potential new targets for antiviral drug development. While the focus of the focus of this project is on the EBOV, the approach taken will be applicable to other related virus-host cell interactions.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.