Structural dynamics of single SARS-CoV-2 pseudoknot molecules reveal topologically distinct conformers.

The RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target. To understand how it responds to mechanical tension applied by ribosomes, thought to play a key role during frameshifting, we probe its structural dynamics using optical tweezers. We find that it forms multiple structures: two pseudoknotted conformers with different stability and barriers, and alternative stem-loop structures. The pseudoknotted conformers have distinct topologies, one threading the 5' end through a 3-helix junction to create a knot-like fold, the other with unthreaded 5' end, consistent with structures observed via cryo-EM and simulations. Refolding of the pseudoknotted conformers starts with stem 1, followed by stem 3 and lastly stem 2; Mg2+ ions are not required, but increase pseudoknot mechanical rigidity and favor formation of the knot-like conformer. These results resolve the SARS-CoV-2 frameshift signal folding mechanism and highlight its conformational heterogeneity, with important implications for structure-based drug-discovery efforts.

Anti-Frameshifting Ligand Active against SARS Coronavirus-2 Is Resistant to Natural Mutations of the Frameshift-Stimulatory Pseudoknot.

SARS-CoV-2 uses -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins. Because modulating -1 PRF can attenuate the virus, ligands binding to the RNA pseudoknot that stimulates -1 PRF may have therapeutic potential. Mutations in the pseudoknot have occurred during the pandemic, but how they affect -1 PRF efficiency and ligand activity is unknown. Studying a panel of six mutations in key regions of the pseudoknot, we found that most did not change -1 PRF levels, even when base-pairing was disrupted, but one led to a striking 3-fold decrease, suggesting SARS-CoV-2 may be less sensitive to -1 PRF modulation than expected. Examining the effects of a small-molecule -1 PRF inhibitor active against SARS-CoV-2, it had a similar effect on all mutants tested, regardless of basal -1 PRF efficiency, indicating that anti-frameshifting activity can be resistant to natural pseudoknot mutations. These results have important implications for therapeutic strategies targeting SARS-CoV-2 through modulation of -1 PRF.