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

Huston, Nicholas C.; Wan, Han; Strine, Madison S.; de Cesaris Araujo Tavares, Rafael; Wilen, Craig; Pyle, Anna Marie

SARS-CoV-2 is the positive-sense RNA virus that causes COVID-19 disease. The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form RNA structures and yet, as much as 97% of its 30 kilobases have not been structurally explored. Here, we apply a novel long amplicon strategy to determine for the first time 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 beta 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.