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

SARS-COV-2病毒是导致COVID19大流行的原因，它通过吸入、附着和进入目标细胞来攻击人体，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，从而捕捉到病毒进入细胞的每一刻。对这一细胞感染阶段的理解依赖于以实际的方式多次记录。在不受传统显微镜技术限制的情况下，具有可视化输入的能力是至关重要的。病毒表面的刺突在细胞插入步骤中起着关键作用。可视化任务需要专门的工具来识别病毒本身(大约是人类头发直径的1/1000)、尖峰(病毒的1/10)和细胞膜的微小特征(与尖峰的尺寸相当)。该项目采用了一种非常适合解决这个问题的新型显微镜--“氦离子显微镜”(HeIM)。COVID19大流行及其可能的继任者代表着一种严重的紧急情况。新的发现和新的调查方法带来了显著的社会效益。该项目将使HEIM成为一项关键技术，用于检查病毒与细胞之间的相互作用，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人员的介绍，为这项新技术的学习和推广到更广泛的病毒学社区提供了一种机制。该项目将使用氦离子显微镜(HEIM)的新成像方式来研究SARS-COV-2病毒与细胞的相互作用，这种成像方式可以识别出优于其他现有技术的形态细节。特别是，这项研究将确定细胞表面的病毒密度，因为众所周知，病毒优先针对特定类型的细胞，如肺部的II型肺细胞。这项研究将量化细胞进入和细胞退出事件的数量，这是细胞表面病毒密度的函数。在此过程中，这些测量将：i)量化病毒细胞进入事件，区分“内吞作用”和“膜融合”；ii)量化病毒细胞退出事件，区分“游离病毒粒子”和“细胞外小泡”释放。这些数据为建立COVID19细胞感染模型和设计治疗方案模型提供了基础。该项目还将侧重于Heim用于优化样品制备、确定临床样品成像程序、开发荧光标记样品的双向相关显微镜等研究，以更好地定义病毒细胞生物化学。这一快速奖项是由分子和细胞生物科学部的分子生物物理学计划颁发的。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.