Review 1: "SARS-CoV-2 escapes direct NK cell killing through Nsp1-mediated downregulation of ligands for NKG2D"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review:

Lee and colleagues perform a rigorous, multi-pronged dissection of the ability of SARS-CoV-2-derived gene products, namely Nsp1, to subvert antiviral functions of natural killer (NK) cells by downregulating ligands for the key activating receptor, NKG2D. This fits nicely into the evolving puzzle of NK cell dysfunction in COVID-19 pathogenesis, in places confirming prior work while refuting some conclusions and shifting the paradigm in others. Specifically, this work confirms and mechanistically expands our understanding that SARS-CoV-2 infection can provoke target cell susceptibility to NK cells. Importantly, this manuscript then adds a temporal component with virus-driven modulation of the surface proteome of infected cells to undermine their recognition by antiviral NK cells.

The approach combines profiling of NK cells after co-incubation with infected cells, a multitude of killing assays including blocking antibodies and longitudinal dissection of virus gene expression, and exploration of the viral proteins involves through single gene expression systems. The data are compelling and impactful for interpretation of how to consider or utilize NK cells in the therapy of SARS-CoV-2 infection. There are some errors in the assembly of the manuscript, including dot plot labels in Figure 1A and a section of the results describing data in Figure 6E&F. Though human data is typically highly variable, the authors are encouraged to explore in their future studies whether the variation among donors observed in most of their figures relates to any predictable features of the donor that would mechanistically impact NK cells responses (such as HLA or KIR haplotype). The use of A549 cells as the platform may limit the generalizable conclusions that can be drawn from the killing of bystander cells, since non-allogeneic non-transformed bystander cells in an infected individual may respond quite distinctly from these targets. The paper is exceptionally well-written with robust citations of the existing literature and a clear depiction of the methods employed. The claims are well-supported by the data and methods used, and decision-makers should consider the claims in this study actionable with very minor reservations about the cell line used in the generation of these, which is notably shared among all of the studies that have probed NK cell responsiveness to SARS-CoV-2 infected cells.