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Review 1: "S373P Mutation of Spike Protein in SARS-CoV-2 Stabilizes Omicron"

Published onSep 12, 2022
Review 1: "S373P Mutation of Spike Protein in SARS-CoV-2 Stabilizes Omicron"

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:

In this study, the authors have detailed an in vitro experiment which measure the mechanical stability of the SARS-CoV-2 spike protein’s receptor binding domain (RBD). An AFM system is used to rapidly pull on individual RBDs, forcing the protein to unfold under force. The authors report that the omicron variant’s RBD shows increased stability compared to pre-omicron RBD as measured by the average force at which the protein domain unfolds. 

While the experiments detailed appear to be well performed and the results are intriguing, the incomplete statistical analysis in the manuscript as currently written make judging the statistical significance of the results difficult. First, there is an inherently large spread of forces for individual unfolding events, with distributions mostly overlapping. The authors report a ~20% difference in unfolding force, but it is not stated if these values are simply averages of all events or the results of fitting Gaussians to the distributions as the figures suggest. Furthermore, the ± values given in the figures (but not explained in the text), which typically report standard error or fitting error, are larger than the difference between variants. Similarly, the authors report an exponential dependence between pulling/loading rate and unfolding force in agreement with a simple energy barrier model (product of force and transition distance). However, the plotted error bars are so large the data could be well fitted with even a constant force value and the fits to the WT and omicron data overlap. As such the extrapolated low force fundamental unfolding rates have fits with errors which exceed the value themselves, making comparisons with any statistical significance impossible. It is not readily apparent if more rigorous analysis of the data can provide more definitive results, if the lack of precision in these measurements is a fundamental result of stochasticity in AFM unfolding measurements, or if the error bars are overestimates that do not represent actual measurement uncertainty.

Finally, while biophysical characterization of biomolecules is publishable, the authors may be overstating the applications of these measurements to viral function and infectivity. It remains to be seen if instability in the beta strands inhibits viral infectivity and would seem counter intuitive unless all other structural elements are more stable including intermolecular interactions such as spike protein binding to ACE2 receptors. Thus, statements such as “This work reveals the stabilizing effect of the S373P mutation and suggests mechanical stability becomes another important factor in SARS-CoV-2 mutation selection” would require correlating these stability measurements with independently acquired measurements of viral infectivity. Overall, we find the manuscript as currently written to be potentially informative, with the opportunity for a stronger conclusion with increased analytical rigor.

We would also like to note that the manuscript references supplementary material that at the time of this review was not posted on BioRxiv with the manuscript. It is possible that material contained in the supplement may address some of the issues mentioned above.


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