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

我们建议通过在单分子水平上的精确力传感实验来研究病毒进入活细胞所必需的分子相互作用。细胞膜是病毒穿透细胞和致病所需克服的主要障碍。作为进入策略的一部分，病毒与细胞表面的特定受体蛋白以一种尚未被很好理解的方式相互作用。这些细胞表面受体通常嵌入细胞膜，在那里它们可以在膜平面上随机移动(通过布朗扩散)。细胞表面受体的物理性质，如它们的移动性和与细胞细胞骨架(细胞膜下的细丝网状结构)的锚定，可能对病毒进入事件产生重要影响。然而，我们目前对这些受体的性质及其在病毒入侵中的作用的了解非常有限。该项目旨在检测和表征病毒受体与细胞细胞骨架之间的分子连接。这些附着物可以通过改变受体的流动性，使受体在细胞表面聚集和/或稳定病毒与受体的相互作用，在病毒入侵过程中发挥关键作用。作为第一步，我们将测量受体-细胞骨架的附着，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线特性，并将为研究这些联系在病毒进入过程中的作用奠定基础。我们将把重点放在人类免疫缺陷病毒(HIV)作为一个模型系统。HIV颗粒首先与免疫系统细胞表面的受体分子CD4和CCR5/CXCR4特异性结合。然后，这些受体在细胞表面的病毒附着部位重新分布和积累。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，HIV的CD4、CCR5和CXCR4受体与细胞细胞骨架之间存在联系。这些联系，加上艾滋病毒附着时细胞骨架的动态重排，被认为是导致艾滋病毒进入所需的受体重新分布和聚集的原因。我们将开发一种新的灵敏仪器(具有纳米、毫秒和亚皮牛顿分辨率)，使我们能够使用光镊子技术结合新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。这一独特的仪器将允许我们提取活细胞膜中的单个CD4受体分子，以确定是否存在由特定连接蛋白建立的CD4和细胞骨架之间的联系。我们将通过比较在细胞表面显示连接蛋白和不连接蛋白的细胞上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进入中所起的作用，这是第一次直接探测假定的CD4-细胞骨架连接。我们的结果将成为未来研究艾滋病毒进入和其他表现出类似进入机制的病毒受体系统的基础。我们的研究可能会为抗病毒药物设计开辟新的途径，为人类健康带来好处，并产生积极的社会和经济影响。此外，在这项研究计划中开发的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。

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.