Live-cell imaging of SARS-CoV-2 replication organelle formation and RNA synthesis

SARS-CoV-2是一种2019年发现的新型b型冠状病毒，导致新冠肺炎疾病，自2019年末以来导致600多万人死亡。虽然最近的病毒学研究已经澄清了SARS-CoV-2如何感染细胞和导致疾病的许多方面，但进入后复制步骤的时空过程仍然存在问题，这可能有助于针对新的治疗方法。利用反向遗传学、活细胞和超分辨显微镜对在细胞中或在SARS-CoV-2感染过程中表达的标记SARS-CoV-2蛋白，我们提出了两个目的，以更好地了解病毒与宿主在感染人呼吸道细胞过程中的相互作用。我们将了解宿主蛋白在SARS-CoV-2诱导的双膜泡(AIM 1)生物发生中的作用，并可视化WT病毒及其变异体(例如Alpha、Delta和Omicron)的SARS-CoV-2 RNA合成(AIM 2)的来源和运输。这些研究将在人类呼吸道上皮细胞系和已鉴定的原代细胞中实时进行。这些目标将通过荧光标记SARS-CoV-2蛋白和病毒RNA以及宿主细胞蛋白来实现。小分子、宿主因子的击倒和病毒基因组中的突变(包括在高循环变种中发现的突变)将被用来改变这些过程，从而研究复制机制。此外，相关的光电子显微镜(CLEM)将提供关于这些复制过程的结构信息。提高对SARS-CoV-2感染的了解可能会为感染患者带来更有效的新冠肺炎疗法，并可能为我们预防或治疗未来出现的新冠状病毒做好准备。

SARS-CoV-2 is a novel b-coronavirus identified in 2019 that causes the disease COVID-19, which is responsible for over 6 million deaths since late 2019. While recent virology studies have clarified many aspects of how SARS- CoV-2 infects cells and causes disease, questions remain on the spatio-temporal processes of post-entry replication steps, which may be useful for targeting novel therapies. Using reverse genetics and live cell and super-resolution microscopy of labeled SARS-CoV-2 proteins expressed in cells or during SARS-CoV-2 infection, we propose two aims to gain better understanding of virus-host interactions during infection of human airway cells. We will understand the role of host proteins in the biogenesis of SARS-CoV-2-induced double- membraned vesicles (Aim 1) and visualize the origin and trafficking of SARS-CoV-2 RNA synthesis (Aim 2) for WT virus and variants of concern (e.g., Alpha, Delta, and Omicron). The studies will be performed in real-time in human airway epithelial cell lines and deidentified primary cells. These aims will be performed by fluorescently labeling SARS-CoV-2 proteins and the viral RNA as well as host cell proteins. Small molecules, knock down of host factors, and mutations in the viral genome (including those found in highly circulating variants) will be used to alter these processes and, thus, infectivity to study replication mechanisms. In addition, correlative light- electron microscopy (CLEM) will provide structural information on these replication processes. Improved understanding of SARS-CoV-2 infection may lead to more effective COVID-19 therapies for infected individuals and could prepare us for preventing or treating new coronaviruses that arise in the future.