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

SARS-CoV-2是2019年发现的一种新型b型冠状病毒，可导致COVID-19疾病，自2019年底以来已造成600多万人死亡。虽然最近的病毒学研究已经阐明了SARS- CoV-2如何感染细胞并导致疾病的许多方面，但对进入后复制步骤的时空过程仍然存在疑问，这可能对靶向新疗法有用。利用反向遗传学、活细胞和超分辨率显微镜观察细胞中或感染期间表达的标记SARS-CoV-2蛋白，我们提出了两个目标，以更好地了解人气道细胞感染期间病毒与宿主的相互作用。我们将了解宿主蛋白在SARS-CoV-2诱导的双膜囊泡（Aim 1）的生物发生中的作用，并可视化WT病毒和相关变异（例如Alpha、Delta和Omicron）的SARS-CoV-2 RNA合成（Aim 2）的起源和运输。该研究将在人气道上皮细胞系和未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、宿主因子的敲除和病毒基因组的突变（包括在高度循环变异中发现的突变）将用于改变这些过程，从而研究复制机制的传染性。此外，相关的光电子显微镜（CLEM）将提供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者提供更有效的COVID-19治疗方法，并为我们预防或治疗未来出现的新型冠状病毒做好准备。

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.