About 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS coronavirus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule–based interventions. All coronaviruses utilize a molecular mechanism called programmed −1 ribosomal frameshift (−1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high −1 PRF rates and that it also harbors a −1 PRF attenuation element. Altering −1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar −1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate −1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit −1 PRF was similarly effective against −1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.

The amplitude of thermodynamic fluctuations in biological macromolecules determines their conformational behavior, dimensions, nature of phase transitions and effectively their specificity and affinity, thus contributing to fine-tuned molecular recognition. Unique among large-scale conformational changes in proteins are temperature-induced collapse transitions in intrinsically disordered proteins (IDPs). Here, we show that CytR DNA-binding domain, an IDP that folds on binding DNA, undergoes a coil-to-globule transition with temperature in the absence of DNA while exhibiting energetically decoupled local and global structural rearrangements, and maximal thermodynamic fluctuations at the optimal bacterial growth temperature. The collapse is shown to be a continuous transition through a combination of statistical-mechanical modeling and all-atom implicit solvent simulations. Surprisingly, CytR binds single-site cognate DNA with negative cooperativity, described by Hill coefficients less than one, resulting in a graded binding response. We show that heterogeneity arising from varying binding-competent CytR conformations or orientations at the single-molecular level contributes to negative binding cooperativity at the level of bulk measurements due to the conflicting requirements of collapse transition, large fluctuations and folding-upon-binding. Our work reports strong evidence for functionally driven thermodynamic fluctuations in determining the extent of collapse and disorder with implications in protein search efficiency of target DNA sites and regulation.

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.

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.

17 years after the SARS-CoV epidemic, the world is facing the COVID-19 pandemic.COVID-19 is caused by a coronavirus named SARS-CoV-2. Given the most optimistic projections estimating that it will take more than a year to develop a vaccine, our best short term strategy may lie in identifying virus-specific targets for small molecule interventions. All coronaviruses utilize a molecular mechanism called -1 PRF to control the relative expression of their proteins. Prior analyses of SARS-CoV revealed that it utilizes a structurally unique three-stemmed mRNA pseudoknot to stimulate high rates of -1 PRF, that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity negatively impacts virus replication, suggesting that this molecular mechanism may be therapeutically targeted. Here we present a comparative analysis of the original SARS-CoV and SARS-CoV-2 frameshift signals. Structural analyses reveal that the core -1 PRF signal, composed of the U UUA AAC slippery site and three-stemmed mRNA pseudoknot is highly conserved. In contrast, the upstream attenuator hairpin is less well conserved. Functional assays revealed that both elements promote similar rates of -1 PRF and that silent coding mutations in the slippery site strongly ablate -1 PRF activity. We suggest that molecules that were previously identified as inhibiting SARS-CoV mediated -1 PRF may serve as lead compounds to counter the current pandemic.