RAPID: What is the role of extracellular vimentin in SARS2 host cell entry?

本项目将确定细胞表面波形蛋白调节新型病毒SARS-CoV-2 （SARS2）的细胞摄取的机制。静脉蛋白是一种中间纤维，构成细胞骨架的一部分。新的证据表明，波形蛋白存在于细胞的细胞外表面，并在多种病毒的结合和摄取中起关键作用；然而，这种情况发生的机制尚不清楚。确定细胞表面波形蛋白的调控因子及其与SARS2的相互作用，将为了解细胞表面波形蛋白的新功能和病毒感染细胞的基本机制提供新的见解。由于我们对这种病毒如何进入细胞的理解不完全，因此迫切需要这些信息。该项目整合了物理学、工程学、化学和生物学的技术，并将通过为研究生和博士后提供跨学科的培训来扩大生物物理学领域的参与。SARS2进入宿主细胞的特征尚未完全确定，但已知抑制内吞作用和直接融合是预防性的。SARS2利用刺突蛋白与血管紧张素转换酶2 （ACE2）细胞受体相互作用，通过膜融合驱动宿主细胞进入。因此，SARS2的膜融合途径与SARS冠状病毒（SARS）相似，SARS冠状病毒在遗传上与SARS2最相似。内吞作用是宿主细胞进入的另一主要途径，但其机制仍不清楚。以往的研究表明，SARS宿主细胞通过内吞作用进入细胞，与细胞表面波形蛋白密切相关。本项目旨在确定细胞表面波形蛋白在SARS2细胞摄取中的作用。本项目旨在实现以下三个目标：1)利用成纤维细胞和肺上皮细胞确定细胞外波形蛋白的存在及其调控因素；2)研究细胞外波形蛋白对SARS2病毒摄取的影响，确定抗波形蛋白抗体能否阻断病毒摄取；3)定量测定vimentin与SARS2刺突蛋白的分子相互作用。这些研究将使用sars病毒样颗粒进行。分子动力学模拟的结果将为实验结果提供一个解释框架。该RAPID奖由分子和细胞生物科学部的细胞动力学和功能项目获得，资金来自《冠状病毒援助、救济和经济安全法案》。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。

This project will determine the mechanisms by which cell surface vimentin modulates the cellular uptake of the novel virus SARS-CoV-2 (SARS2). Vimentin is an intermediate filament that forms part of the cellular cytoskeleton. New evidence indicates that vimentin is present on the extracellular surface of cells and plays a critical role in the binding and uptake of multiple viruses; yet, the mechanisms by which this happens remain unclear. Identifying the factors that regulate cell surface vimentin and how it interacts with SARS2 will provide new insight into new functions of cell surface vimentin and the fundamental mechanisms by which viruses infect cells. There is an urgent need for this information as we have an incomplete understanding of how this virus enters the cell. This project integrates techniques from physics, engineering, chemistry, and biology and will broaden participation in the field of biophysics by providing interdisciplinary training for graduate students and postdocs.Host cell entry of SARS2 is not yet fully characterized, but inhibition of both endocytosis and direct fusion are known to be preventative. SARS2 makes use of a spike protein to interact with the angiotensin converting enzyme 2 (ACE2) cellular receptor to drive host cell entry via membrane fusion. The membrane fusion pathway for SARS2 is, therefore, similar to SARS-CoV (SARS), the virus that genetically most closely resembles SARS2. Endocytosis is another dominant pathway for host cell entry, but the mechanisms involved remains largely unknown. Previous studies indicate that SARS host cell entry via endocytosis critically involves cell surface vimentin. This project aims to determine the role of cell surface vimentin in SARS2 cellular uptake. This project pursues the following three objectives: 1) determine the presence and the factors that regulate extracellular vimentin using fibroblasts and lung epithelial cells; 2) study the effect of extracellular vimentin on SARS2 viral uptake and determine whether viral uptake can be blocked by anti-vimentin antibodies; and 3) quantify the molecular interaction between vimentin and the spike protein of SARS2. These studies will be conducted using SARS2 virus-like particles. Results from molecular dynamics simulations will provide an interpretative framework for the experimental results.This RAPID award is made by the Cellular Dynamics and Functionl Program in the Division of Molecular and Cellular Biosciences, using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act.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.