Review 3: "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 study, the authors identified single nucleotide variants of the SARS-CoV-2 Nsp15 by comparing around 1.9 million full-length sequences of SARS-CoV-2 present in the GISAID database (as of June 2021) with the sequence of the original Wuhan isolate. By analyzing this large dataset that represents global isolates, the authors note that Nsp15 has nucleotide substitutions at 1025 positions out of its total 1038 nucleotides. However, most of these variations (87 %) are detected in less than 100 isolates, and only 83 nucleotide variations (around 8 % of the total) are present in more than 1000 isolates. Further, only 53 out of the 83 variations led to amino acid changes, the other 30 are synonymous. There is only one non-synonymous mutation (D220Y) in more than 10,000 isolates and 5 synonymous variants. By following the presence of non-synonymous substitutions, the authors in this report provide additional evidence for the independent origin of the VOCs, Delta and Omicron.

Further, the authors selected seventeen of the non-synonymous variants for biochemical analysis, which involved examination of protein oligomerization and RNA cleavage activity by using assays reported in earlier studies of Nsp15. Nsp15 is a uridine-specific endoribonuclease, which is conserved among coronaviruses and plays a role in the evasion of the innate immune response. Additionally, Nsp15 is suggested to serve as a scaffold in the assembly of viral Replication Transcription Complex (RTC), play a role in virus transmission and is shown to interact with the cellular protein, retinoblastoma protein (pRb). Discussion and references are provided for the role of Nsp15 in evasion of the innate immune response, involvement in RTC formation, and virus transmission but not for interaction with pRb. 

Biochemical analysis performed in this study has identified new residues that are involved in the stabilization of Nsp15 hexamer and a residue in the N-terminal domain that might be involved in RNA binding. Although, it remains to be demonstrated through direct RNA binding assays. Intriguingly, variant V128F showed 2-fold higher activity whereas variants R207S and K260R showed significantly low activity, but the mechanism remains to be examined. 

Earlier studies on SARS CoV Nsp15 have shown that substitution of certain residues on this protein renders the virus non-viable, making it a suitable antiviral target. Therefore, understanding the mechanistic details of Nsp15 is important. However, most of the biochemical analysis provided in this study has only re-confirmed what is already known about the endoribonuclease activity of Nsp15. Characterization of the additional variants where conserved residues are altered or the variants with low number of isolates and correlation with the patient outcome (if the data is available) could provide useful insight. It is worth noting that the residues which are altered in more than 1000 isolates (supplemental file 1) are not highly conserved among coronaviruses and are therefore tolerated. Strikingly, many variants with less than 10 isolates contain stop codons/premature protein.

In addition, the amino acid numbering in the text is off by one and therefore does not match with the amino acid numbering in supplemental file 1. Figure 3C should be mentioned in the text.