ABSTRACT
SARS-CoV-2 carries the largest single-stranded RNA genome and is the causal pathogen of the ongoing COVID-19 pandemic. How the SARS-CoV-2 RNA genome is folded in the virion remains unknown. To fill the knowledge gap and facilitate structure-based drug development, we developed a virion RNA in situ conformation sequencing technology, named vRIC-seq, for probing viral RNA genome structure unbiasedly. Using vRIC-seq data, we reconstructed the tertiary structure of the SARS-CoV-2 genome and revealed a surprisingly "unentangled globule" conformation. We uncovered many long-range duplexes and higher-order junctions, both of which were under purifying selections and contributed to the sequential package of the SARS-CoV-2 genome. Unexpectedly, the D614G and the other two accompanying mutations might remodel duplexes into more stable forms. Lastly, the structure-guided design of potent small interfering RNAs could obliterate the SARS-CoV-2 in Vero cells. Overall, our work provides a framework for studying the genome structure, function, and dynamics of emerging deadly RNA viruses.