A cytosine-to-uracil change within the programmed -1 ribosomal frameshift signal of SARS-CoV-2 results in structural similarities with the MERS-CoV signal

Fourmy, Dominique; Yoshizawa, Satoko

doi:10.1101/2020.06.26.174193

Cited by 9 publications

(38 citation statements)

References 58 publications

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“…The urgency of the COVID-19 pandemic, recent advances in targeting RNA 3D structures with ASOs and small molecules, and the identification of the FSE as a potentially well-defined RNA 3D structure in the SARS-CoV-2 genome has generated significant interest in understanding and targeting SARS-CoV-2 ribosomal frameshifting 3,23,27,28,44,45,50,51 . Here, we confirmed the ability of ASOs to invade the SARS-CoV-2 FSE structure and to reduce frameshifting efficiencies in cellfree assays, and we explored the potential for these ASOs to reduce viral replication in cells at submicromolar concentrations.…”

Section: Discussionmentioning

confidence: 99%

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

Zhang

Zheludev

Hagey

et al. 2020

Preprint

135

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Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome. The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides. To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 Å, presents a topologically complex fold in which the 5′ end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.

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Section: Discussionmentioning

confidence: 99%

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

Zhang

Zheludev

Hagey

et al. 2020

Preprint

135

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“…Understanding the impact of mutations is vital to long-term success of treatments that are based on predicted RNA structures. Interestingly, the most prevalent mutation to the SARS-CoV-2 −1 PRF pseudoknot sequence was found to increase the similarity of the SARS-CoV-2 −1 PRF pseudoknot structure to that of the MERS-CoV −1 PRF pseudoknot structure [26]. Further validation of the similarity between the SARS-CoV-2 and MERS-CoV −1 PRF pseudoknots is highly relevant for future coronavirus structure prediction efforts.…”

Section: Introductionmentioning

confidence: 94%

“…In addition, mutations have been reported in all components of the SARS-CoV-2 −1 PRF pseudoknot [26,27]. Yet, the possible effects of such mutations on the RNA structure formation and resulting secondary structures have not been fully investigated.…”

Section: Introductionmentioning

confidence: 99%

“…In order to follow up on the hypothesized link between the SARS-CoV-2 and MERS-CoV −1 PRF pseudoknot structures [26], we enumerate the specific loci of similarity as well as a consensus structure. Where previous work has identified the importance of non-native pseudoknotted structures, our research goes further by explicitly identifying non-native structures and predicting how the structure is realized.…”

Section: Introductionmentioning

confidence: 99%

“…Mutations were reported in nearly every base in the SARS-CoV-2 −1 PRF pseudoknot sequences [27] available on GenBank [43] and GISAID [44]. A single nucleotide change from cytosine to uracil at position 62 (C62U) has been identified in an increasing proportion of sequences, up to 3% since the mutation was first recorded in March 2020 [26]. Due to the complicated nature of RNA folding in vivo, even a single nucleotide change can affect the resulting structure.…”

Section: Introductionmentioning

confidence: 99%

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SARS-CoV-2 ribosomal frameshifting pseudoknot: Improved secondary structure prediction and detection of inter-viral structural similarity

Trinity

Lansing

Jabbari

et al. 2020

Preprint

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the COVID-19 pandemic; a pandemic of a scale that has not been seen in the modern era. Despite over 29 million reported cases and over 900, 000 deaths worldwide as of September 2020, herd immunity and widespread vaccination efforts by many experts are expected to be insufficient in addressing this crisis for the foreseeable future. Thus, there is an urgent need for treatments that can lessen the effects of SARS-CoV-2 in patients who become seriously affected. Many viruses including HIV, the common cold, SARS-CoV and SARS-CoV-2 use a unique mechanism known as −1 programmed ribosomal frameshifting (−1 PRF) to successfully replicate and infect cells in the human host. SARS-CoV (the coronavirus responsible for SARS) and SARS-CoV-2 possess a unique RNA structure, a three-stemmed pseudoknot, that stimulates −1 PRF. Recent experiments identified that small molecules can be introduced as antiviral agents to bind with the pseudoknot and disrupt its stimulation of −1 PRF. If successfully developed, small molecule therapy that targets −1 PRF in SARS-CoV-2 is an excellent strategy to improve patients’ prognoses. Crucial to developing these successful therapies is modeling the structure of the SARS-CoV-2 −1 PRF pseudoknot. Following a structural alignment approach, we identify similarities in the −1 PRF pseudoknots of the novel coronavirus SARS-CoV-2, the original SARS-CoV, as well as a third coronavirus: MERS-CoV, the coronavirus responsible for Middle East Respiratory Syndrome (MERS). In addition, we provide a better understanding of the SARS-CoV-2 −1 PRF pseudoknot by comprehensively investigating the structural landscape using a hierarchical folding approach. Since understanding the impact of mutations is vital to long-term success of treatments that are based on predicted RNA functional structures, we provide insight on SARS-CoV-2 −1 PRF pseudoknot sequence mutations and their effect on the resulting structure and its function.

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Tying the Knot: Unraveling the Intricacies of the Coronavirus Frameshift Pseudoknot

Trinity,

Stege,

Jabbari

2023

Preprint

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Understanding and targeting functional RNA structures towards treatment of coronavirus infection can help us to prepare for novel variants of SARS-CoV-2 (the virus causing COVID-19), and any other coronaviruses that could emerge via human-to-human transmission or potential zoonotic (inter-species) events. Leveraging the fact that all coronaviruses use a mechanism known as -1 programmed ribosomal frameshifting (-1 PRF) to replicate, we apply algorithms to predict the most energetically favourable secondary structures (each nucleotide involved in at most one pairing) that may be involved in regulating the -1 PRF event in coronaviruses, especially SARS-CoV-2. We compute previously unknown most stable structure predictions for the frameshift site of coronaviruses via hierarchical folding, a biologically motivated framework where initial non-crossing structure folds first, followed by subsequent, possibly crossing (pseudoknotted), structures. Using mutual information from 181 coronavirus sequences, in conjunction with the algorithm KnotAli, we compute secondary structure predictions for the frameshift site of different coronaviruses. We then utilize the Shapify algorithm to obtain most stable SARS-CoV-2 secondary structure predictions guided by frameshift sequence-specific and genome-wide experimental data. We build on our previous secondary structure investigation of the singular SARS-CoV-2 68 nt frameshift element sequence, by using Shapify to obtain predictions for 132 extended sequences and including covariation information. Previous investigations have not applied hierarchical folding to extended length SARS-CoV-2 frameshift sequences. By doing so, we simulate the effects of ribosome interaction with the frameshift site, providing insight to biological function. We contribute in-depth discussion to contextualize secondary structure dual-graph motifs for SARS-CoV-2, highlighting the energetic stability of the previously identified 3_8 motif alongside the known dominant 3_3 and 3_6 (native-type) -1 PRF structures. Integrating experimental data within minimum free energy (MFE) hierarchical folding algorithms provides novel structure predictions to distill the relationship between RNA structure and function. In particular, fully categorizing most stable secondary structure predictions via hierarchical folding supports our identification of motif transitions and critical site targets for future therapeutic research.

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A cytosine-to-uracil change within the programmed -1 ribosomal frameshift signal of SARS-CoV-2 results in structural similarities with the MERS-CoV signal

Cited by 9 publications

References 58 publications

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

SARS-CoV-2 ribosomal frameshifting pseudoknot: Improved secondary structure prediction and detection of inter-viral structural similarity

Tying the Knot: Unraveling the Intricacies of the Coronavirus Frameshift Pseudoknot

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