Length-dependent motions of SARS-CoV-2 frameshifting RNA pseudoknot and alternative conformations suggest avenues for frameshifting suppression

Conserved SARS-CoV-2 RNA regions of critical biological functions define excellent targets for anti-viral therapeutics against Covid-19 variants. One such region is the frameshifting element (FSE), responsible for correct translation of viral polyproteins. Here, we analyze molecular-dynamics motions of three FSE conformations, discovered by graph-theory analysis, and associated mutants designed by graph-based inverse folding: two distinct 3-stem H-type pseudoknots and a 3-way junction. We find that the prevalent H-type pseudoknot in literature adopts ring-like conformations, which in combination with 5′ end threading could promote ribosomal pausing. An inherent shape switch from “L” to linear that may help trigger the frameshifting is suppressed in our designed mutant. The alternative conformation trajectories suggest a stable intermediate structure with mixed stem interactions of all three conformations, pointing to a possible transition pathway during ribosomal translation. These observations provide new insights into anti-viral strategies and frameshifting mechanisms.

Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch

Programmed –1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.

Crystal structure of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frameshifting pseudoknot

SARS-CoV-2 produces two long viral protein precursors from one open reading frame using a highly conserved RNA pseudoknot that enhances programmed -1 ribosomal frameshifting. The 1.3 Å-resolution X-ray structure of the pseudoknot reveals three coaxially stacked helices buttressed by idiosyncratic base triples from loop residues. This structure represents a frameshift-stimulating state that must be deformed by the ribosome, and exhibits base-triple-adjacent pockets that could be targeted by future small-molecule therapeutics.

A Novel Frameshifting Inhibitor Having Antiviral Activity against Zoonotic Coronaviruses

Recent outbreaks of zoonotic coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have caused tremendous casualties and great economic shock. Although some repurposed drugs have shown potential therapeutic efficacy in clinical trials, specific therapeutic agents targeting coronaviruses have not yet been developed. During coronavirus replication, a replicase gene cluster, including RNA-dependent RNA polymerase (RdRp), is alternatively translated via a process called -1 programmed ribosomal frameshift (−1 PRF) by an RNA pseudoknot structure encoded in viral RNAs. The coronavirus frameshifting has been identified previously as a target for antiviral therapy. In this study, the frameshifting efficiencies of MERS-CoV, SARS-CoV and SARS-CoV-2 were determined using an in vitro −1 PRF assay system. Our group has searched approximately 9689 small molecules to identify potential −1 PRF inhibitors. Herein, we found that a novel compound, 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline (KCB261770), inhibits the frameshifting of MERS-CoV and effectively suppresses viral propagation in MERS-CoV-infected cells. The inhibitory effects of 87 derivatives of furo[2,3-b]quinolines were also examined showing less prominent inhibitory effect when compared to compound KCB261770. We demonstrated that KCB261770 inhibits the frameshifting without suppressing cap-dependent translation. Furthermore, this compound was able to inhibit the frameshifting, to some extent, of SARS-CoV and SARS-CoV-2. Therefore, the novel compound 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline may serve as a promising drug candidate to interfere with pan-coronavirus frameshifting.

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.

Restriction of SARS-CoV-2 replication by targeting programmed −1 ribosomal frameshifting

Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires a programmed −1 ribosomal frameshift (−1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in −1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a −1 PRF inhibitor for SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on −1 PRF of other betacoronaviruses. Consistent with the essential role of −1 PRF in viral gene expression, merafloxacin impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing proof-of-principle for targeting −1 PRF as a plausible and effective antiviral strategy for SARS-CoV-2 and other coronaviruses.

Salt-Dependent RNA Pseudoknot Stability: Effect of Spatial Confinement

Macromolecules, such as RNAs, reside in crowded cell environments, which could strongly affect the folded structures and stability of RNAs. The emergence of RNA-driven phase separation in biology further stresses the potential functional roles of molecular crowding. In this work, we employed the coarse-grained model that was previously developed by us to predict 3D structures and stability of the mouse mammary tumor virus (MMTV) pseudoknot under different spatial confinements over a wide range of salt concentrations. The results show that spatial confinements can not only enhance the compactness and stability of MMTV pseudoknot structures but also weaken the dependence of the RNA structure compactness and stability on salt concentration. Based on our microscopic analyses, we found that the effect of spatial confinement on the salt-dependent RNA pseudoknot stability mainly comes through the spatial suppression of extended conformations, which are prevalent in the partially/fully unfolded states, especially at low ion concentrations. Furthermore, our comprehensive analyses revealed that the thermally unfolding pathway of the pseudoknot can be significantly modulated by spatial confinements, since the intermediate states with more extended conformations would loss favor when spatial confinements are introduced.