Some of these conformers involve knot-like fold topologies that have not previously been observed in frameshift-stimulatory pseudoknots, specifically conformers with the 5′ end threaded through the junction between the three helices to generate what we term a ring-knot 10, 15, 18. Cryo-EM imaging 10, 15 and computational modeling 18 both suggest that the SARS-CoV-2 pseudoknot can take on several different conformers (Fig. 1a) that is characteristic of coronaviruses, in contrast to the more common two-stem architecture of most viral frameshift-stimulatory pseudoknots 17. The pseudoknot stimulating −1 PRF in SARS-CoV-2 has a three-stem architecture 1, 10, 15, 16 (Fig. As a result, the structures stimulating −1 PRF are potential targets for anti-viral drugs 7, 8, 9, motivating efforts to find ligands active against −1 PRF in SARS-CoV-2 that could be used to treat COVID-19 10, 11, 12, 13, 14.
Previous work on viruses including HIV-1 and SARS-CoV showed that mutations modulating the level of −1 PRF can significantly attenuate viral propagation in cell culture 4, 5, 6. In −1 PRF, a shift in the reading frame of the ribosome at a specific location in the RNA message is stimulated by a structure in the mRNA located 5–7 nt downstream of the slippery sequence where the reading frameshift occurs, thereby generating alternate gene products 2, 3. Like most coronaviruses, the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) causing the COVID-19 pandemic makes use of −1 programmed ribosomal frameshifting (−1 PRF) to express proteins that are essential for viral replication 1. 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. Refolding of the pseudoknotted conformers starts with stem 1, followed by stem 3 and lastly stem 2 Mg 2+ ions are not required, but increase pseudoknot mechanical rigidity and favor formation of the knot-like conformer.
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. We find that it forms multiple structures: two pseudoknotted conformers with different stability and barriers, and alternative stem-loop structures. 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. The RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target.