Direct probing of molecular interactions relevant to virus entry via force spectroscopy with optical tweezers in live cells

使用光镊在活细胞中通过力谱直接探测与病毒进入相关的分子相互作用

• 批准号: EP/P020747/1
• 负责人: Isabel Llorente Garcia
• 金额: $ 11.6万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FP020747%2F1
• 关键词: Direct; probing; molecular; interactions; relevant

We propose to investigate the molecular interactions necessary for virus entry into living cells by means of precision force-sensing experiments at the single molecule level. The cell membrane is the main barrier that viruses need to overcome to penetrate cells and cause disease. As part of their entry strategy, viruses interact with specific receptor proteins at the cell surface in ways which are not well understood. These cell-surface receptors are typically embedded in the membrane of the cell, where they can move randomly (via Brownian diffusion) in the membrane plane. The physical properties of cell-surface receptors, such as their mobility and anchoring to the cellular cytoskeleton (a mesh of filaments beneath the cell membrane), are likely to importantly influence virus entry events. However, our knowledge of these receptor properties and their role in virus entry is currently very limited.This project aims at detecting and characterising molecular attachments between virus receptors and the cellular cytoskeleton. These attachments can play a crucial role in virus entry by modifying receptor mobility, enabling the clustering of receptors on the cell surface and/or stabilising virus-receptor interactions. As a first step, we will measure receptor-cytoskeleton attachments to determine if they are present in the absence of viruses. This will allow us to understand the baseline properties of virus receptors and will set a basis upon which to investigate the role of these links during virus entry.We will focus on the Human Immunodeficiency Virus (HIV) as a model system. HIV particles first attach specifically to receptor molecules CD4 and CCR5/CXCR4 on the surface of cells of the immune system. These receptors then redistribute and accumulate at the sites of virus attachment on the cell surface. Eventually, the virus penetrates the cell membrane and releases its genome into the cellular cytoplasm. Several recent studies have pointed towards links between the CD4, CCR5 and CXCR4 receptors for HIV and the cellular cytoskeleton. These links, together with dynamic rearrangements of the cytoskeleton upon HIV attachment, have been suggested as responsible for the receptor redistribution and clustering required for HIV entry. However, the proposed links have not been observed directly to date and the mechanisms for clustering remain unknown.We will develop a new sensitive instrument (with nanometre, millisecond and sub-picoNewton resolution) that will allow us to measure these unknown interactions at the cell surface using optical tweezer technology combined with novel sequential data acquisition and real-time data analysis. This unique instrument will allow us to pull individual CD4 receptor molecules in the membrane of living cells to establish whether connections made by specific linker proteins exist between CD4 and the cytoskeleton. We will do this by comparing force measurements on cells displaying CD4 on their surface with and without the linker proteins. Our results will enable us to understand the role that receptor-cytoskeleton interactions play in virus entry, with this being the first time that putative CD4-cytoskeleton attachments are probed directly. Our results will form the basis of future investigations into HIV entry and into other virus-receptor systems that exhibit similar entry mechanisms. Our research will potentially open new avenues for anti-viral drug design, generating benefits to human health and positive societal and economic impact. Furthermore, the techniques developed in this research programme for measuring and characterising molecular interactions will be broadly applicable to various biomedical and biophysical problems that involve cell-surface receptors and are important to human health such as, for instance, cell growth in cancer and immune response to infections.

我们建议通过单分子水平的精确力传感实验来研究病毒进入活细胞所需的分子相互作用。细胞膜是病毒穿透细胞并致病所需要克服的主要屏障。作为其进入策略的一部分，病毒与细胞表面的特定受体蛋白相互作用的方式尚不清楚。这些细胞表面受体通常嵌入细胞膜中，它们可以在细胞膜平面上随机移动（通过布朗扩散）。细胞表面受体的物理特性，例如它们的移动性和锚定在细胞骨架（细胞膜下的细丝网）上，可能会对病毒进入事件产生重要影响。然而，我们对这些受体特性及其在病毒侵入中的作用的了解目前非常有限。该项目旨在检测和表征病毒受体和细胞骨架之间的分子附着。这些附着物可以通过改变受体的移动性，使受体在细胞表面聚集和/或稳定病毒与受体的相互作用，在病毒进入中发挥关键作用。作为第一步，我们将测量受体-细胞骨架附着物，以确定它们是否在没有病毒的情况下存在。这将使我们能够了解病毒受体的基本特性，并为研究这些链接在病毒进入过程中的作用奠定基础。我们将重点关注人类免疫缺陷病毒（HIV）作为一个模型系统。HIV颗粒首先特异性地附着在免疫系统细胞表面的受体分子CD4和CCR5/CXCR4上。然后这些受体重新分布并聚集在病毒附着在细胞表面的位置。最终，病毒穿透细胞膜，将其基因组释放到细胞质中。最近的几项研究指出了HIV的CD4、CCR5和CXCR4受体与细胞骨架之间的联系。这些连接，连同细胞骨架在HIV附着时的动态重排，被认为是HIV进入所需的受体重新分布和聚集的原因。然而，到目前为止，所提出的联系还没有直接观察到，聚类的机制仍然未知。我们将开发一种新的敏感仪器（具有纳米，毫秒和亚皮牛顿分辨率），这将使我们能够使用光学镊子技术结合新颖的顺序数据采集和实时数据分析来测量细胞表面的这些未知相互作用。这种独特的仪器将使我们能够在活细胞膜中提取单个CD4受体分子，以确定CD4和细胞骨架之间是否存在由特定连接蛋白建立的连接。我们将通过比较在有和没有连接蛋白的情况下在细胞表面显示CD4的细胞的力测量来做到这一点。我们的结果将使我们能够理解受体-细胞骨架相互作用在病毒侵入中所起的作用，这是第一次直接探测假定的cd4 -细胞骨架附着物。我们的结果将为未来研究HIV进入和其他表现出类似进入机制的病毒受体系统奠定基础。我们的研究将有可能为抗病毒药物设计开辟新的途径，为人类健康和积极的社会和经济影响带来好处。此外，在本研究方案中开发的用于测量和表征分子相互作用的技术将广泛适用于涉及细胞表面受体的各种生物医学和生物物理问题，这些问题对人类健康很重要，例如，癌症中的细胞生长和对感染的免疫反应。