Visualization of early RNA replication kinetics of SARS-CoV-2 by using single molecule RNA-FISH.
Rajiv PathakCarolina EliscovichIgnacio MenaUpdesh DixitAdolfo García-SastreRobert H SingerGanjam V KalpanaPublished in: bioRxiv : the preprint server for biology (2022)
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.
Keyphrases
- single molecule
- sars cov
- single cell
- living cells
- atomic force microscopy
- cell death
- rna seq
- dna damage
- respiratory syndrome coronavirus
- reactive oxygen species
- cell therapy
- palliative care
- induced apoptosis
- cell cycle arrest
- computed tomography
- stem cells
- risk factors
- bone marrow
- cell proliferation
- dna methylation
- endoplasmic reticulum stress
- case control