Abstract: The formation of double membrane vesicles (DMVs), a critical step in coronavirus infection to shield viral genomes from innate immune receptors, necessitates the involvement of various host factors. Previous research has identified several host proteins, including TMEM41B, VMP1, and RTN3/4, which facilitate the interaction between viral non-structural proteins Nsp3 and Nsp4, ER zippering and curving, and the fusion of paired ER membranes necessary for DMV biogenesis. Despite these findings, there remains a lack of high-throughput methods for identifying host factors involved in DMV formation. In this study, we employ a genome-wide CRISPR-knockout/activation library screening to identify host factors that either promote or suppress the interaction between Nsp3 and Nsp4. Our validation results indicate that the knockdown or overexpression of specific host factors can modulate the strength of the Nsp3-Nsp4 interaction, influencing DMV formation, as well as viral replication and kinetics in SARS-CoV-2-infected human lung carcinoma cell lines. Having these preliminary findings, we extend our study to human pluripotent stem cell-derived airway epithelium, providing a more physiologically relevant model of DMV biogenesis in true infection scenarios, with implications for antiviral strategies.
