ABSTRACT
SARS-CoV-2 infection has caused a major global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood including the kinetics of formation of plus stranded genomic and subgenomic RNAs (gRNA and sgRNA) starting from the RNA from the first virus that enters the cell. We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA in infected cells and carried out a time course analysis to determine the kinetics of their replication. We visualized the single molecules of gRNA within the cytoplasm of infected cells 30 minutes post-infection and detected the co-expression of gRNA and sgRNA within two hours post-infection. Furthermore, we observed the formation of a replication organelle (RO) from a single RNA, which led to the formation of multiple ROs within the same cells. Single molecule analysis indicated that while gRNA resided in the center of these ROs, the sgRNAs were found to radiate and migrate out of these structures. Our results also indicated that after the initial delay, there was a rapid but asynchronous replication, and the gRNA and sgRNAs dispersed throughout the cell within 4-5 hours post-infection forming multiple ROs that filled the entire cytoplasm. These results provide insight into the kinetics of early post-entry events of SARS-CoV-2 and the formation of RO, which will help to understand the molecular events associated with viral infection and facilitate the identification of new therapeutic targets that can curb the virus at a very early stage of replication to combat COVID-19.