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

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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.