RNA viruses possess strictly conserved RNA segments that adopt local structures and act as regulatory cis-acting elements. In addition, long-range RNA-RNA interactions between distant parts of their genome provide additional layers of regulatory control. In SARS‑CoV‑2, crosslinking experiments identified multiple long-range interactions within the genome, including the longest distant interaction between nucleotides in the 5’- and the 3’‑terminal, untranslated regions (UTR’s). Formation of this interaction requires opening of the 5’SL3 element in the 5’-UTR, which contains the transcription regulatory core sequence (TRS‑L), and of the 3’SL3base element in the 3’-UTR. Here, we investigate the minimal sequence context required for formation of this long-range interaction. We determine the alternative secondary structures adopted by these elements, their thermodynamic stability, and the stability of individual base pairs. Furthermore, we quantify equilibrium populations and exchange kinetics between the circularization duplex and the alternative local structures. We investigate duplex formation with larger, native-like UTR constructs, determining populations similar to those of the minimal constructs. Finally, we demonstrate that circularization is substantially less favorable in subgenomic RNAs than in the genomic RNA, suggesting that genome circularization may contribute to discrimination between functions involving either genomic or subgenomic RNAs during the viral life cycle.

raw NMR and MST data and a script to fit EXSY data from the manuscript and the Supporting Information