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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.

Published onJul 21, 2021
Review 2: "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:

  • Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review:

This manuscript is potentially informative, but several controls need to be done and there are other possible interpretations of some of the data.  The authors claim that eIF4A enhances cap accessibility based on the data that eIF4E binding is increased in the presence of additional eIF4A.  This is an interesting result, but it is not evident that it is due to increased cap accessibility.  Several of the studies with eIF4A and ATP should be repeated with a non-hydrolyzable ATP analog to determine the effects—if any—of helicase reactions.  All of these studies would be more believable if a non-functional cap were used—such as ApppA—to determine if the reactions are truly with the mRNA cap or possibly with other structures in the 5’ UTR. EIF 4G, for example, is known to interact with RNA secondary structures. It seems likely that SARS-CoV translation is more eIF4A-dependent than some mRNA and this is an interesting avenue to pursue.  It is also well known that cap-binding depends on the nearby RNA structures, so this finding is not surprising. Two additional controls would provide more confidence in the conclusions. One is to measure ensemble binding to the mRNA to eIF4E when the mRNA is not immobilized.  While the ensemble binding would not provide the same information as the single-molecule measurements, this data should be similar in overall affinity if the binding is not perturbed by the immobilization.  Along these same lines, the authors do not state how they refolded the RNA oligonucleotides, which is likely important in binding and translation. Lastly, the authors labeled the eIF4E and added a small peptide extension.  It is not shown whether or not this modification alters cap-binding or translation.  This should be compared with wild-type eIF4E.  Overall, there are some potentially intriguing results that should be further developed.

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