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.