The RNA genomes of coronaviruses comprise many conserved structures and distal RNA-RNA interactions that play essential roles in the viral replication cycle. In the 3’ untranslated region of betacoronaviruses, a pseudoknot RNA structure 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. On the basis of bioinformatic analyses and a mutagenesis approach combined 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 contact with an exposed sequence in the coding region of the SARS-CoV-2 genome¹. The bases involved in this contact are highly conserved in all SARS-like coronaviruses and sequencing results indicate that this motif may form an alternative polymerase binding site, enabling the synthesis of non-canonical subgenomic RNA. These findings contribute information about the molecular mechanisms governing the processes of replication, discontinuous transcription and recombination in these viruses, and may expand the scope of therapeutic strategies against current or future infections by sarbecoviruses.

(1) López-Núñez, S., Cantero-Camacho, A., Simba-Lahuasi, A., Ye, C., Mena, I., García-Sastre, A., Martínez-Sobrido, L., Gallego, J. A potential RNA-RNA distal interaction involving a functional pseudoknot in the 3'-untranslated region of SARS-like coronaviruses. Submitted (2025).