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Review 2: "Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants"

This preprint aims to characterize the impact of SARS-CoV-2 Nsp15 variants on its oligomerization state and nuclease activity. Reviewers find the study informative, with scope for improvement in the analysis of the oligomerization state.

Published onJun 07, 2022
Review 2: "Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants"
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key-enterThis Pub is a Review of
Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants
Description

AbstractGlobal sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9+ million SARS-CoV-2 sequences revealed mutations across Nsp15’s three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants in vivo.

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:

Wilson et al. biochemically characterized different SARS-CoV-2 Nsp15 variants, which they extracted from the GISAID database of SARS-CoV-2 sequences. They analyzed selected mutations regarding their oligomerization state and nuclease activity. In general, I agree with the statement, that it is important to biochemically prove the effect of different mutations on the protein function and not only predict the impact based on structural data. However, the analysis of the oligomerization state should have been done with similar protein concentrations. In Figures 3b, 4b, and 5b this was clearly not the case. All curves should have a similar integrated area under all peaks. At least in the pdf version, the different curves are partially invisible. A better method for oligomerization analysis would be massphotometry. The structural figures could have been prepared more carefully e.g. color-coded labeling, and better close-up views (the active site is basically invisible in Figure 2). In general, most close-up figures are uninformative (Fig. 3A,4A, 5A).

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