Sarbecovirus programmed ribosome frameshift RNA element folding studied by NMR spectroscopy and comparative analyses.

The programmed ribosomal frameshift (PRF) region is found in the RNA genome of all coronaviruses and shifts the ribosome reading frame through formation of a three-stem pseudoknot structure, allowing the translation of essential viral proteins. Using NMR spectroscopy, comparative sequence analyses and functional assays we show that, in the absence of the ribosome, a 123-nucleotide sequence encompassing the PRF element of SARS-CoV-2 adopts a well-defined two-stem loop structure that is conserved in all SARS-like coronaviruses. In this conformation, the attenuator hairpin and slippery site nucleotides are exposed in the first stem-loop and two pseudoknot stems are present in the second stem-loop, separated by an 8-nucleotide bulge. Formation of the third pseudoknot stem depends on pairing between bulge nucleotides and base-paired nucleotides of the upstream stem-loop, as shown by a PRF construct where residues of the upstream stem were removed, which formed the pseudoknot structure and had increased frameshifting activity in a dual-luciferase assay. The base-pair switch driving PRF pseudoknot folding was found to be conserved in several human non-SARS coronaviruses. The collective results suggest that the frameshifting pseudoknot structure of these viruses only forms transiently in the presence of the translating ribosome. These findings clarify the frameshifting mechanism in coronaviruses and can have a beneficial impact on antiviral drug discovery.