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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.

Published onJul 21, 2021
Review 1: "Single-Molecule Dynamics of SARS-CoV-2 5ʹ Cap Recognition by Human eIF4F"
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Single-Molecule Dynamics of SARS-CoV-2 5’ Cap Recognition by Human eIF4F
Description

ABSTRACTCoronaviruses initiate translation through recognition of the viral RNA 5’ m7GpppAm 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.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review:

Although the subject (COVID-19 mRNA) is of extreme interest due to the current worldwide pandemic, this reviewer does not feel that there is sufficiently new material presented to justify publication — even though many of the experiments appear to have been done well and with controls.  Other laboratories have examined many of the interactions of the eIF4F subunits with mRNA and this is acknowledged in this article.  In addition, several concerns about the experimental methodology are raised.

Concerns:

1.      Regarding the fluorescently-labeled eIF4E, it is not clear if the labeling is random (to more than one site on eIF4E or targeted to a specific amino acid) and whether this might influence results.  Secondly, if about 50% is labeled, does the unmodified eIF4E serve as a “competitive” inhibitor?

2.     Regarding the eIF4A ATPase assay, do the authors know that the rate of oxidation of NADH is much faster than the possible rate of ATP hydrolysis?

3.     In the results, the authors wrote: “Thus, the viral untranslated regions support eIF4F-dependent translation”.  Is this true for both UTRs or just the 5’ UTR?

4.     For experiments with more than a single subunit, do the authors have proof of the degree with which complexes are formed (i.e. eIF4G + eIF4E —> eIF4G•4E)?  Secondly, what are the kinetics for the formation of complexes, especially eIF4F, when individual subunits are added together for the assay? 

5.     The authors used proteins expressed individually, although the eIF4G is not full-length.  How does the observed eIF4F activity compare to native eIF4F?

6.     For the presumed stem-loops in the COVID-19 5’ UTR, what is the calculated ∆G of the stems?

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