In vivo structure and dynamics of the RNA genome of SARS-Cov-2

The SARS-CoV-2 coronavirus, which causes the COVID-19 pandemic, is one of the largest positive strand RNA viruses. Here we developed a simplified SPLASH assay and comprehensively mapped the in vivo RNA-RNA interactome of SARS-CoV-2 RNA during the viral life cycle. We observed canonical and alternative structures including 3'-UTR and 5'-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in cells and in virions. We provide the first evidence of comprehensive interactions between Transcription Regulating Sequences (TRS-L and TRS-Bs), which facilitate discontinuous transcription. In addition, we find alternative short and long distance arches around FSE, forming a “high-order pseudoknot” embedding FSE, which might help ribosome stalling at frameshift sites. We found that during packaging, SARS-CoV-2 genome RNA undergoes compaction while genome domains remain stable and genome cyclization is weakened. Our data provides a structural basis for the regulation of replication, discontinuous transcription and translational frameshifting describes dynamics on RNA structures during life cycle of SARS-CoV-2, and will help to develop antiviral strategies. Overall design: We developed a simplified SPLASH protocol to capture RNA-RNA interactions in the SARS-CoV-2 virus. Briefly, the biological samples are first stabilized by Psoralen-PEG3-Biotin cross-linking, followed by RNase III treatment, proximity ligation, library preparation, and sequencing. Samples were collected from different phases of the SARS-CoV-2 virus viral cycle to infer the dynamic structure of viral RNA at different stages. In the early stage of infection, we collected virus-infected Vero cells (C), in which cytopathic effect (CPE) was not observed. In the later stage of infection, when 70% cells underwent CPE, we collected the cell culture supernatant and harvested virus particles (V) and, at the same time used freeze-thaw methods to lyse the cells (L) to collect the cell and virus RNA