Enhanced-sampling simulations reveal distinct intermediates in SARS-CoV-2 FSE pseudoknot interconversion

The frameshifting stimulatory element (FSE) of SARS-CoV-2 regulates programmed -1 ribosomal frameshifting, a process critical for viral protein synthesis, which has garnered attention as a potential antiviral target. In SARS-CoV-2, a 3-stem H-type pseudoknot has been widely recognized as the primary structure stimulating -1 frameshifting. However, both experimental and computational studies have revealed that the FSE is structurally heterogeneous and can adopt alternative structures. While this conformational plasticity is believed to play a central role in modulating frameshifting efficiency, transition pathways between these alternative FSE structures have remained poorly understood. Here, we use Multithermal-Multiumbrella On-the-Fly Probability Enhanced Sampling technique at atomistic resolution to sample interconversions between two H-type 3-stem pseudoknots of the 87-nucleotide SARS-CoV-2 FSE, known as motifs 3_3 and 3_6, respectively. Reweighted free-energy projections identify well-defined basins corresponding to both motifs, together with intermediate regions associated with partial loss and reorganization of motif-specific contacts. Analysis of enhanced-sampling interconversion trajectories reveals distinct terminal-segment-mediated contacts in the two endpoint-initiated simulations. Trajectories initiated from 3_3 sample both stepwise routes, in which stem 2 of 3_3 unfolds before stem 2 of 3_6 forms, and more concurrent routes, in which loss of 3_3 contacts overlaps with formation of 3_6 contacts and transient triplex-like interactions involving the 3' end. In contrast, trajectories initiated from 3_6 predominantly sample stepwise rearrangements among successful transitions, while some trajectories populate metastable intermediates stabilized by nonnative contacts between the 5' end and stem 2 of 3_6. These results identify structural intermediates and contact rearrangements that may contribute to the conformational plasticity of the SARS-CoV-2 FSE.