A potential RNA-RNA distal interaction competing with a functional pseudoknot in the 3'-untranslated region of SARS-like coronaviruses.

In the 3' untranslated region of betacoronaviruses, a pseudoknot involving stem-loop 3SL2 has been proposed to form a binding site for the viral RNA polymerase enabling the initiation of negative-sense RNA synthesis during the processes of virus replication and discontinuous transcription. Based on bioinformatic analyses and a mutagenesis strategy monitored with electrophoretic experiments and viral replication assays, we report that, in addition to the local pseudoknot in the 3'UTR, 3SL2 can establish a distal tertiary contact with an exposed sequence in the ORF7b gene located 1847 nucleotides upstream in the SARS-CoV-2 genome. The bases involved in this contact are highly conserved in all SARS-like coronaviruses and sequencing data indicate that this motif may form an alternative polymerase binding site enabling the synthesis of non-canonical subgenomic RNA. These findings provide a foundation for investigating the molecular mechanisms governing the processes of replication, discontinuous transcription, and recombination in these viruses.