To Knot or Not to Knot: Multiple Conformations of the SARS-CoV-2 Frameshifting RNA Element

Abstract

The SARS-CoV-2 frameshifting RNA element (FSE) is an excellent target for therapeutic intervention against Covid-19. This small gene element employs a shifting mechanism to pause and backtrack the ribosome during translation between Open Reading Frames 1a and 1b, which code for viral polyproteins. Any interference with this process has a profound effect on viral replication and propagation. Pinpointing the structures adapted by the FSE and associated structural transformations involved in frameshifting has been a challenge. Using our graph-theory-based modeling tools for representing RNA secondary structures, “RAG” (RNA-As-Graphs), and chemical structure probing experiments, we show that the 3-stem H-type pseudoknot (3_6 dual graph), long assumed to be the dominant structure, has a viable alternative, an HL-type 3-stem pseudoknot (3_3) for longer constructs. In addition, an unknotted 3-way junction RNA (3_5) emerges as a minor conformation. These three conformations share Stems 1 and 3, while the different Stem 2 may be involved in a conformational switch and possibly associations with the ribosome during translation. For full-length genomes, a stem-loop motif (2_2) may compete with these forms. These structural and mechanistic insights advance our understanding of the SARS-CoV-2 frameshifting process and concomitant virus life cycle, and point to three avenues of therapeutic intervention.

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

“…1 ). This motif has been reported by chemical probing, Cryo-EM, NMR, crystallography 3 , 16 – 22 , and molecular dynamics (MD) 23 – 25 . Using our coarse-grained RNA-As-Graphs (RAG) representation as dual graphs 26 – 29 , we assign this pseudoknot motif dual graph 3_6 (Fig.…”

“…The functional importance and high conservation of the FSE make it a promising candidate for anti-viral drugs and gene therapy; for example, in the latest Omicron variant, there are 31 new mutations in the spike gene region with respect to the previous variants, but no changes in the FSE (Supplementary Fig. 1) 3 – 6 . Whether frameshifting is orchestrated by the FSE acting as a road blocker or through more complex conformational switches remains unknown 7 – 13 .…”

“…Using our coarse-grained RNA-As-Graphs (RAG) representation as dual graphs 26 – 29 , we assign this pseudoknot motif dual graph 3_6 (Fig. 1 ) 3 , 24 . RAG translates double-stranded stems to vertices and single-stranded loops to edges.…”

Targeting Ribosomal Frameshifting as an Antiviral Strategy: From HIV-1 to SARS-CoV-2

“…Likewise, recent X-ray crystallographic data supports the existence of the pseudoknot . Structure probing data combined with graph theoretical analysis on the SARS-CoV-2 genomic RNA in vitro and structure probing inside virions, however, suggests the frameshift element may exist either as an H-type pseudoknot, an HL-type pseudoknot, a 3-way junction, or a large stem–loop. These results suggest that further research is required to determine whether the secondary structure depends on context or a dynamic equilibrium exists.…”

Translational Control of COVID-19 and Its Therapeutic Implication

“…Repressor of yield of dengue virus (RyDEN) is induced after a SARS-CoV-2 infection, binds to SARS-CoV-2-RNA in infected cells and regulates the efficiency of SARS-CoV-2 -1 PRF ( 96 ). In addition, the SARS-CoV-2 FSE is functionally obligate for viral fitness, suggesting that the stimulatory element could be used as a therapeutic target ( 87 , 93 , 97 ). Sun et al.…”