Structural dynamics of the SARS-CoV-2 frameshift-stimulatory pseudoknot reveal topologically distinct conformers (preprint)

The RNA pseudoknot that stimulates -1 programmed ribosomal frameshifting in SARS coronavirus-2 (SARS-CoV-2) is a possible drug target. To understand how this 3-stemmed pseudoknot responds to the mechanical tension applied by ribosomes during translation, which is thought to play a key role during frameshifting, we probed its structural dynamics under tension using optical tweezers. Unfolding curves revealed that the frameshift signal formed multiple different structures: at least two distinct pseudoknotted conformers with different unfolding forces and energy barriers, as well as alternative stem-loop structures. Refolding curves showed that stem 1 formed first in the pseudoknotted conformers, followed by stem 3 and then stem 2. By extending the handle holding the RNA to occlude the 5' end of stem 1, the proportion of the different pseudoknot conformers could be altered systematically, consistent with structures observed in cryo-EM images and computational simulations that had distinct topologies: the 5' end of the RNA threaded through the 3-helix junction to form a ring-knot, or unthreaded as in more standard H-type pseudoknots. These results resolve the folding mechanism of the frameshift signal in SARS-CoV-2 and highlight the dynamic conformational heterogeneity of this RNA, 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 (preprint)

The coronavirus SARS-CoV-2 causing the COVID-19 pandemic uses −1 programmed ribosomal frameshifting (−1 PRF) to control the expression levels of key viral proteins. Because modulating −1 PRF can attenuate viral propagation, ligands binding to the viral RNA pseudoknot that stimulates −1 PRF may prove useful as therapeutics. Mutations in the pseudoknot have been observed over the course of the pandemic, but how they affect −1 PRF and the activity of inhibitors is unknown. Cataloguing natural mutations in all parts of the SARS-CoV-2 pseudoknot, we studied a panel of 6 mutations in key structural regions. Most mutations left the −1 PRF efficiency unchanged, even when base-pairing was disrupted, but one led to a remarkable three-fold decrease, suggesting that SARS-CoV-2 propagation may be less sensitive to modulation of −1 PRF efficiency than some other viruses. Examining the effects of one of the few small-molecule ligands known to suppress −1 PRF significantly in SARS-CoV, we found that it did so by similar amounts in all SARS-CoV-2 mutants tested, regardless of the basal −1 PRF efficiency, indicating that the activity of anti-frameshifting ligands can be resistant to natural pseudoknot mutations. These results have important implications for therapeutic strategies targeting SARS-CoV-2 through modulation of −1 PRF.Competing Interest StatementThe authors have declared no competing interest.View Full Text