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.
In this article, the authors describe the biochemical characterization of Nsp15 variants extracted from the GISAID database of SARS-CoV-2 sequences acquired during the pandemic to understand how mutations found in SARS-CoV-2 variants would impact Nsp15 function. Among many nonstructural proteins of SARS-CoV-2 Nsp15 has been understudied compared to PLpro (Nsp3), Mpro (Nsp5), and RNA dependent RNA Polymerase (RdRp, Nsp12). Biochemical and in vivo studies of SARS-CoV-2 protein variants beyond the spike protein could contribute to a fuller picture of how the coronavirus proteome functions which should provide insight into drug design.
Nuclease activity of Nsp15 helps the virus evade the MDA5 mediated innate immune response. The hexameric structure of the uridine-specific endonuclease Nsp15 is well-known and shown to have an oligomerization-dependent nuclease activity: the monomeric form is much less active than the hexamer. The authors specifically analyzed changes in oligomerization state and nuclease activity.
They selected mutations from each of three domains: the N-terminal domain (NTD) has been shown to be critical for oligomerization, the middle domain (MD) plays an important role in stabilizing the hexamer, and the C-terminal EndoU domain with catalytic/active site residues. They looked at the frequency of mutation and active site residues utilizing the wealth of bioinformatics data accumulated for SARS CoV-2 (1.9+ million sequences) and carried out size exclusion chromatography and gel electrophoresis. Combined with the previously reported studies including the structural data, they tried to provide a conceivable explanation.
They focused on the nonsynonymous variants and found many highly prevalent variants tend to have decreased nuclease activity with increased monomeric forms. The correlation between hexamer formation and nuclease activities of various mutants was explained through a structural basis.
Among many mutations studied, they identified several mutants of large impact but couldn’t be expected based on the correlation between hexamer formation and nuclease activities. For example, for the T34I mutation and in the N-terminal domain, the oligomeric state is not perturbed, however, nuclease assays reveal a significant decrease in activity. Similarly, for V128F, the residue is not directly involved in any protomer interfaces particularly far from the EndoU site but the assays indicated the mutation decreases the formation of the hexamer. However, the hexamer formed has a substantial increase in nuclease activity.
Also, the H235Y stands out as a candidate to further study in viruses as one of catalytic residue, found in so many isolates and characterized as a lineage marker for a clade of the Delta variant, Delta D.
Also speculated that variants that do not affect oligomerization or activity nonetheless could affect RNA packaging by referring to the work to develop virus-like particles to study SARS-CoV-2 variants found that an important RNA packaging signal overlaps with the Nsp15 coding sequence suggesting that synonymous amino acid mutations may also result in changes to RNA packaging, which may not be related to the oligomerization of Nsp15.
Relating to the atomistic model of hexameric Nsp15 being the scaffold for a hexameric RTC based on computational modeling tools, the catalytic residues of H235Y and K290N being dead mutation while increasing the hexamer formation is explained. H235 and K290 are the catalytic residues and are expected to critically impact catalysis but how these residues can increase hexamer formation (compared to WT) has never been explained clearly.
Several curves in the size exclusion chromatography (SEC) chromatograms are not well resolved and bit difficult to follow.
Within the limited scope of biochemical in vitro characterization, good pieces of work but could have been more useful if the cellular study is included which seems out of scope.