Evolution of coronavirus frameshifting elements: Competing stem networks explain conservation and variability

Abstract

The frameshifting RNA element (FSE) in coronaviruses (CoVs) regulates the programmed −1 ribosomal frameshift (−1 PRF) mechanism common to many viruses. The FSE is of particular interest as a promising drug candidate. Its associated pseudoknot or stem loop structure is thought to play a large role in frameshifting and thus viral protein production. To investigate the FSE structural evolution, we use our graph theory-based methods for representing RNA secondary structures in the RNA-As-Graphs (RAG) framework to calculate conformational landscapes of viral FSEs with increasing sequence lengths for representative 10 Alpha and 13 Beta-CoVs. By following length-dependent conformational changes, we show that FSE sequences encode many possible competing stems which in turn favor certain FSE topologies, including a variety of pseudoknots, stem loops, and junctions. We explain alternative competing stems and topological FSE changes by recurring patterns of mutations. At the same time, FSE topology robustness can be understood by shifted stems within different sequence contexts and base pair coevolution. We further propose that the topology changes reflected by length-dependent conformations contribute to tuning the frameshifting efficiency. Our work provides tools to analyze virus sequence/structure correlations, explains how sequence and FSE structure have evolved for CoVs, and provides insights into potential mutations for therapeutic applications against a broad spectrum of CoV FSEs by targeting key sequence/structural transitions.

Abolished frameshifting for predicted structure-stabilizing SARS-CoV-2 mutants: Implications to alternative conformations and their statistical structural analyses

“…These data are also consistent with other groups’ analyses of the FSE secondary structure (Lan et al 2022; Pekarek et al 2023; Yan et al 2022; Rangan et al 2021; Schlick, Zhu, Dey, et al 2021). We have emphasized in our prior work that not only can the RNA fold into different conformations for the same length (Schlick, Zhu, Dey, et al 2021; Yan et al 2023; Schlick, Zhu, Jain, et al 2021); the fold distribution is also highly sensitive to length, when residues are either added or deleted (Yan et al 2023) .…”

“…As a control we also DMS probed, modeled, and measured the frameshifting efficiency of two additional longer WT constructs (156nt and 222nt long) (Figures 4A, B, and C, respectively). Our previous work has shown that longer constructs of SARS-CoV-2 FSE tend to adopt other conformations due to a competing Stem 1 and attenuator hairpin (Yan et al 2022; Yan et al 2023) (S. lee, S. Yan, A. Dey et al, Pers. comm., 2024).…”

“…As discussed in this work, the SARS-CoV-2 FSE not only plays a critical role in coronavirus replication and virulence (K. Zhang et al 2021; Sanders et al 2020; Iserman et al 2020; Schlick, Zhu, Jain, et al 2021; Schlick, Zhu, Dey, et al 2021; Yan et al 2022; Yan et al 2023; Jones and Ferré-D’Amaré 2022; Bhatt et al 2021; Roman et al 2021), but is also of intense interest because of the intriguing repertoire of length dependent conformations. Prior studies have reported many possible conformations (K. Zhang et al 2021; Bhatt et al 2021; Jones and Ferré-D’Amaré 2022; Roman et al 2021; Manfredonia et al 2020; Iserman et al 2020; Sanders et al 2020; Schlick, Zhu, Dey, et al 2021) for the SARS-CoV-2 FSE, although cryo-EM and crystallographic studies have identified the H-type pseudoknot (3_6) at short lengths (K. Zhang et al 2021; Jones and Ferré-D’Amaré 2022; Roman et al 2021).…”

Tertiary folds of the SL5 RNA from the 5′ proximal region of SARS-CoV-2 and related coronaviruses

“…Among viral RNA structures, previous comparisons, conducted by NMR or X-ray crystallography, were limited to the comparison of two mutants (67) or two homologs (68)(69)(70). Structural comparisons for coronavirus RNA genomes have been limited to RNA secondary structure, including analysis of the 5' UTR (47,57) and the frameshift stimulation element (71), rather than comparisons of tertiary structure. Cryogenic electron microscopy (cryo-EM) offers the possibility of more routinely characterizing RNA elements across multiple homologs, particularly when integrated with biochemical secondary structure determination and automated computer modeling, such as in the Ribosolve pipeline (72).…”

NMR Studies of Genomic RNA in 3′ Untranslated Region Unveil Pseudoknot Structure that Initiates Viral RNA Replication in SARS-CoV-2

“…8 A pseudoknot structure is a structure motif of nucleic acids that has two (or more) stem-loops; in a basic pseudoknot structure, one stem is nested into the loop region of the other stem-loop. Pseudoknot structures were found in structured RNAs involved in important biological processes, such as the frameshift element of viruses, 13,14 the telomerase RNA component, 15 and tRNAlike structures. 16 Nonstructural proteins (nsp)7−16, which are coded in the ORF1ab gene at the 5′ terminus of the genome, play primary roles in replicating the virus genome.…”