RAPID / SARS-CoV-2 host cell interactions: quantitative investigations via Scanning Helium-ion Microscopy

RAPID / SARS-CoV-2 宿主细胞相互作用：通过扫描氦离子显微镜进行定量研究

• 批准号: 2115363
• 负责人: Leonard Feldman
• 金额: $ 19.36万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2115363&HistoricalAwards=false
• 关键词: RAPID; SARS; CoV; host; cell

The SARS-COV-2 virus, the cause of the COVID19 pandemic, attacks the human body by inhalation, attachment on and entry into a target cell, followed by virus multiplication and, in the most severe cases, disruption of the immune system. This research will reveal images of viruses, while they are attacking the outside of a cell, similar to an observer flying a helicopter on a battlefield. At the same time, it will be possible to zoom-in so powerfully that the very moment of the virus entry into the cell will be captured. The understanding of this cell-infecting stage depends on documenting it multiple times and in a practical way. It is critical to have the ability to visualize the entry without the limitations of traditional microscopy techniques. Spikes on the surface of the virus play a critical role in the cell insertion step. The visualization task requires specialized tools able to identify the virus itself (approximately 1/1000 the diameter of a human hair), the spike (1/10 of the virus), and tiny features of the cell membrane (comparable to the dimension of the spike). This project employs a new microscope ideally suited to this problem, a “Helium-ion Microscope” (HeIM). The COVID19 pandemic, and its possible successors, represent an acute emergency. New discoveries and new methods of investigation provide a significant social benefit. This project will establish HeIM as a technique critical to examine the virus-cell interaction for possible future needs. Involvement of students and young scientists in the research, together with its introduction to investigators of viral diseases, provide a mechanism for this new technology to be learned and spread to the broader virology community.The project will study the SARS-COV-2 virus-cell interaction by using the new imaging modality of Helium-ion Microscopy (HeIM) which allows identification of morphological details superior to other established techniques. In particular, the study will determine the viral density on the cell surface, as it is known that the virus preferentially targets specific cell types, like the Type II Pneumocytes of the lungs. The study will quantify the number of cell-entry and cell-exit events that occur as a function of viral density on the cell surface. In so doing, these measurements will: i) quantify viral cell-entry events, differentiating between “endocytosis” and “membrane fusion” and ii) quantify viral cell-exit events, differentiating between “free-virion” and “extracellular vesicle” release. Such data provides a basis for COVID19 cell infection modeling and for designing models of therapeutic options. The project will also focus on HeIM use for such studies of optimizing sample preparation, determining procedures for imaging clinical samples, developing bi-directional correlative microscopy with fluorescent-labelled samples which better define the virus-cell biochemistry. This RAPID award is made by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences.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.

引起COVID-19大流行的SARS-COV-2病毒通过吸入、附着和进入靶细胞攻击人体，随后病毒繁殖，在最严重的情况下，破坏免疫系统。这项研究将揭示病毒在攻击细胞外部时的图像，类似于观察者在战场上驾驶直升机。与此同时，它将有可能如此强大地放大，以至于病毒进入细胞的那一刻将被捕获。对这个细胞感染阶段的理解取决于多次以实用的方式记录它。关键是要有能力可视化的进入，而不受传统显微镜技术的限制。病毒表面的刺突在细胞插入步骤中起关键作用。可视化任务需要能够识别病毒本身（约为人类头发直径的1/1000），刺突（病毒的1/10）和细胞膜的微小特征（与刺突的尺寸相当）的专用工具。该项目采用了一种新的显微镜，非常适合这个问题，“氦离子显微镜”（HeIM）。COVID-19大流行及其可能的后继者代表着一种严重的紧急情况。新的发现和新的调查方法提供了重大的社会效益。该项目将建立HeIM作为一种关键技术，用于检查病毒-细胞相互作用，以满足未来可能的需求。学生和年轻科学家的参与，以及对病毒疾病研究者的介绍，为这一新技术的学习和传播提供了一个机制。该项目将利用氦离子显微镜（HeIM）的新成像模式研究SARS-COV-2病毒与细胞的相互作用，该成像模式可以识别上级其他现有技术的形态细节。特别是，该研究将确定细胞表面上的病毒密度，因为已知病毒优先靶向特定的细胞类型，如肺部的II型肺细胞。该研究将量化细胞进入和细胞退出事件的数量，这些事件作为细胞表面上病毒密度的函数发生。在这样做时，这些测量将：i）定量病毒细胞进入事件，区分“内吞作用”和“膜融合”，和ii）定量病毒细胞离开事件，区分“游离病毒体”和“细胞外囊泡”释放。这些数据为COVID 19细胞感染建模和设计治疗方案模型提供了基础。该项目还将重点关注HeIM用于优化样品制备，确定临床样品成像程序，开发具有荧光标记样品的双向相关显微镜，以更好地定义病毒细胞生物化学。该奖项由分子和细胞生物科学部的分子生物物理计划颁发。该奖项反映了NSF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。