Review 4: "Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants"

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:

In this report, the authors reported the biochemical characterization of emerging SARS-CoV-2 Nsp15 uridine-specific endoribonuclease variants identified from a large group of patients. Bioinformatic analyses of more than 1.9 million SARS-CoV-2 Nsp15 sequences revealed mutations across Nsp15’s three structured domains (amino-terminal domain (NTD), middle domain (MD), and catalytic endoribonuclease (EndoU) domain). Moreover, the structural analyses of the mutations identified in the variants using the currently available three-dimensional structure models of the enzyme provided significant insight into the roles of the mutations in the function of the endoribonuclease. Selected variants were expressed in E.coli. These variant proteins were purified and biochemically characterized for their enzymatic activities to cleave the mRNA substrates and their ability to assemble into a functional complex. The enzymatic activity was assayed using both FRET and gel-based cleavage assays. The oligomerization of the variants was studied using gel-filtration assays. 

The conclusion of the report is important and has provided valuable information to our understanding of SARS-CoV-2 encoded endoribonuclease. For example, the bioinformatic analyses revealed that the evolution of the endoribonuclease is independent from the emergence of some of the most prevalent variants, such as the delta and omicron variants of the spike protein, found in the human population during the pandemic. This result is interesting but not entirely unexpected, suggesting that at the very least, the endoribonuclease and the spike protein are under different and independent selection pressures to mutate and adapt. 

Another interesting and important finding of the report is the identification of variants that contain mutations in the active site of the EndoU domain, almost completely inactivating enzymatic activity. These observations strongly suggest that the ribonuclease activity of Nsp15 may not be essential for SARS-CoV-2 and COVID because the variants were isolated from infected individuals in vivo. These results also raise the possibility that Nsp15 may have other functions that are essential for viral infection and pathogenesis. While the authors mentioned the recent proposed computational model and hypothesis in which Nsp15 may serve as a scaffold for the assembly of the Replication Transcription Complex, experimental evidence is needed to determine if this is the case.  

The approaches used in the report represent a wonderful combination of bioinformatic analyses, structural modeling, and biochemical characterization. The experiments are well conceived and clearly described. The conclusions of the manuscript are supported by the results. I only have several comments and minor concerns listed below:

1. The authors nicely analyzed the nature of the variants in NTD, MD, and EndoU domains, and their ability to cleave the RNA substrates and to form a complex. The results showed that there were no correlations between the enzymatic activity of the Nsp15 variants and their levels of prevalence in human populations in vivo. However, the nature of mutations in the spike proteins in these Nsp15 variant SARS-CoV-2 strains is not presented. Additional analyses on the association between the Nsp15 mutations and the mutations from other viral genes (e.g. S and E proteins) within the same viral strains are important. The presentation of these analyses would greatly improve the manuscript. 

2. Additional biochemical characterization may further improve the manuscript and provide insight into the effect of the mutations on the activity of Nsp15. For example, the authors may consider further assaying the binding affinity of the enzyme to the RNA substrate, given the fact that they have purified a selected collection of variant proteins. 

3. Little discussion is included to discuss possible sequence errors in the database they used for the bioinformatic analysis. For example, what were the rates of sequencing error? This discussion may be needed in the case of the variants with the mutations at the active site that abolished the endoribonuclease activity. It is true that the mutated enzymes were completely inactive in the authors’ assays. However, it is possible that the mutated enzyme was not present in vivo and the mutations found in the database were due to sequencing error. 

4. Some statements may need to be revised for clarity. For example, several sentences in the third paragraph of the Result section discussing the MD variants may need to be restructured or moved to the Discussion section. The results indicated that the enzymatic activity of Nsp15 variants increased and decreased compared to the Nsp15 of the original strain. No results were presented about the infection and replication of the viruses carrying the Nsp15 variants. So it is more appropriate not to discuss the correlation between the level of enzymatic activity in these variants and the level of the proliferation and infection of the viruses carrying these variants.