Visualization of early RNA replication kinetics of SARS-CoV-2 by using single molecule RNA-FISH.

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 Author Summary: SARS-CoV-2 infection continues to be a global burden. Soon after the entry, SARS-CoV-2 replicates by an elaborate process, producing genomic and subgenomic RNAs (gRNA and sgRNAs) within specialized structures called replication organelles (RO). Many questions including the timing of multiplication of gRNA and sgRNA, the generation, subcellular localization, and function of the ROs, and the mechanism of vRNA synthesis within ROs is not completely understood. Here, we have developed probes and methods to simultaneously detect the viral gRNA and a sgRNA at single cell single molecule resolution and have employed a method to scan thousands of cells to visualize the early kinetics of gRNA and sgRNA synthesis soon after the viral entry into the cell. Our results reveal that the replication is asynchronous and ROs are rapidly formed from a single RNA that enters the cell within 2 hours, which multiply to fill the entire cell cytoplasm within ~4 hours after infection. Furthermore, our studies provide a first glimpse of the gRNA and sgRNA synthesis within ROs at single molecule resolution. Our studies may facilitate the development of drugs that inhibit the virus at the earliest possible stages of replication to minimize the pathogenic impact of viral infection.