Single-Molecule Dynamics of SARS-CoV-2 5’ Cap Recognition by Human eIF4F
This article has been Reviewed by the following groups
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- Evaluated articles (ScreenIT)
- Evaluated articles (Rapid Reviews Infectious Diseases)
Abstract
Coronaviruses initiate translation through recognition of the viral RNA 5’ m 7 GpppA m cap by translation factor eIF4F. eIF4F is a heterotrimeric protein complex with cap-binding, RNA-binding, and RNA helicase activities. Modulating eIF4F function through cellular regulation or small-molecule inhibition impacts coronavirus replication, including for SARS-CoV-2. Translation initiation involves highly coordinated dynamics of translation factors with messenger or viral RNA. However, how the eIF4F subunits coordinate on the initiation timescale to define cap-binding efficiency remains incompletely understood. Here we report that translation supported by the SARS-CoV-2 5’-UTR is highly sensitive to eIF4A inhibition by rocaglamide. Through a single-molecule fluorescence approach that reports on eIF4E–cap interaction, we dissect how eIF4F subunits contribute to cap-recognition efficiency on the SARS-CoV-2 5’ UTR. We find that free eIF4A enhances cap accessibility for eIF4E binding, but eIF4G alone does not change the kinetics of eIF4E–RNA interaction. Conversely, formation of the full eIF4F complex significantly alters eIF4E–cap interaction, suggesting that coordinated eIF4E and eIF4A activities establish the net eIF4F–cap recognition efficiency. Moreover, the eIF4F complex formed with phosphomimetic eIF4E(S209D) binds the viral UTR more efficiently than with wild-type eIF4E. These results highlight a dynamic interplay of eIF4F subunits and mRNA that determines cap-recognition efficiency.
Article activity feed
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Dixie Goss
Review 2: "Single-Molecule Dynamics of SARS-CoV-2 5ʹ Cap Recognition by Human eIF4F"
This preprint uses a single molecular assay to demonstrate that SARS-CoV-2 5’ translation is sensitive to the small molecule rocaglamide. Reviewers deem the methodology reliable with only minor follow-up experiments.
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Review 1: "Single-Molecule Dynamics of SARS-CoV-2 5ʹ Cap Recognition by Human eIF4F"
This preprint uses a single molecular assay to demonstrate that SARS-CoV-2 5’ translation is sensitive to the small molecule rocaglamide. Reviewers deem the methodology reliable with only minor follow-up experiments.
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Strength of evidence
Reviewers: 📗📗📗📗◻️
Dixie Goss (CUNY) | 📒📒📒◻️◻️ -
SciScore for 10.1101/2021.05.26.445185: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis Movies were acquired for 10 minutes at 10 frames per second, under illumination with a 532 nm laser at a power of 0.70 µW/µm2, to excite Cy3 fluorophores. Cell Line Authentication Authentication: 5 Traces containing robust smFRET signals for eIF4E–RNA interaction were selected by visual inspection. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Luciferase assays: 100 nM CoV firefly luciferase fusion RNA was added to 12 µL of HeLa lysate reaction mix from an in vitro protein expression kit (Thermo Scientific, 88882), along with 0.5 µL of rocaglamide/RocA solution … SciScore for 10.1101/2021.05.26.445185: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis Movies were acquired for 10 minutes at 10 frames per second, under illumination with a 532 nm laser at a power of 0.70 µW/µm2, to excite Cy3 fluorophores. Cell Line Authentication Authentication: 5 Traces containing robust smFRET signals for eIF4E–RNA interaction were selected by visual inspection. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Luciferase assays: 100 nM CoV firefly luciferase fusion RNA was added to 12 µL of HeLa lysate reaction mix from an in vitro protein expression kit (Thermo Scientific, 88882), along with 0.5 µL of rocaglamide/RocA solution (Med Chem Express, Product No. HY-19356) or 4E2RCat solution (Med Chem Express, Product No. HY-100733) in varying concentrations as indicated in the Results and Discussion. HeLasuggested: NoneRecombinant DNA Sentences Resources For preparation of the luciferase fusion construct, DNA fragments containing the class II T7 promoter Φ2.5, followed by the SARS-CoV-2 5’ untranslated region sequence, the coding sequence for firefly luciferase, and the SARS-CoV-2 5’ untranslated region sequence were assembled in a pUC57 vector. pUC57suggested: RRID:Addgene_40306)The sequence was inserted into pUC119 via SalI and EcoRI restriction sites incorportated during gBlock synthesis. pUC119suggested: RRID:Addgene_50010)Preparation of fluorescently labeled eIF4E and eIF4E(S209D): The pET-28a(+) vectors containing the sequences of eIF4E or eIF4E(S209D), each fused with an N-terminal Protein G tag, were designed similarly to Feokistova et. al., (2013). pET-28a(+ )suggested: NoneThe pULTRA expression vector, containing inserts encoding tRNACUA and pAzF-tRNACUA-synthetase – a generous gift from Abhishek Chatterjee, Boston College – was co-transformed into an E. coli BL95ΔAΔfabR strain with the pET28a(+)-eIF4E plasmid, and transformants were selected on LB agar containing Kanamycin (50 µg/mL) and Spectinomycin (100 µg/mL). pULTRAsuggested: RRID:Addgene_24129)pET28a(+)-eIF4Esuggested: NoneSoftware and Algorithms Sentences Resources The full-length human GAPDH transcript sequence, obtained from UCSC Genome Browser, was purchased as a gBlock from IDT. UCSC Genome Browsersuggested: (UCSC Genome Browser, RRID:SCR_005780)48 Event timings identified from ebFRET analysis were converted to empirical cumulative probability distributions for the times between events, and the event durations, then fit with single- or double-exponential models to determine kon or koff, according to the equations: Fitting was carried out in MATLAB by non-linear least-squares regression using the Trust-Region algorithm.
MATLABsuggested: (MATLAB, RRID:SCR_001622)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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