Comprehensive in vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms

Severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2) is the positive-sense RNA virus that causes coronavirus disease 2019 (COVID-19). The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form RNA structures, yet as much as 97% of its 30 kilobases have not been structurally explored. Here, we apply a novel long amplicon strategy to determine the secondary structure of the SARS-CoV-2 RNA genome at single-nucleotide resolution in infected cells. Our in-depth structural analysis reveals networks of well-folded RNA structures throughout Orf1ab and reveals aspects of SARS-CoV-2 genome architecture that distinguish it from other RNA viruses. Evolutionary analysis shows that several features of the SARS-CoV-2 genomic structure are conserved across β-coronaviruses, and we pinpoint regions of well-folded RNA structure that merit downstream functional analysis. The native, secondary structure of SARS-CoV-2 presented here is a roadmap that will facilitate focused studies on the viral life cycle, facilitate primer design, and guide the identification of RNA drug targets against COVID-19. [Display omitted] •The SARS-CoV-2 genome is probed at single-nucleotide resolution in infected cells•RNA structure prediction reveals an elaborate SARS-CoV-2 genome architecture•Networks of well-folded secondary structure are conserved across β-coronaviruses•Disruption of conserved secondary structures with LNAs inhibits viral growth Using cell-based chemical probing, Huston et al. provide an experimentally determined structural map of the SARS-CoV-2 RNA genome in infected cells. The map reveals networks of well-folded RNA structures that are conserved in other coronaviruses. Disruption of these structures inhibits SARS-CoV-2 growth.