Review 2: "Emergence of Transmissible SARS-CoV-2 Variants with Decreased Sensitivity to Antivirals in Immunocompromised Patients with Persistent Infections"

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 study and characterize the emergence of resistance mutations (during persistent infections) in immunocompromised COVID-19 patients to both nirmatrelvir and remdesivir. Using in-vivo studies in golden Syrian hamsters, the authors also demonstrate that these mutations are transmissible.

The study is reasonably well designed and the conclusions are in general supported by the evidence provided. The manuscript is also well written. I have a few comments regarding key results:

1. Structural analyses of the nsp5 T169I and nsp12 V792I mutations revealed that while these mutations are not in the corresponding binding pockets of nirmatrelvir or remdesivir, these mutations lead to significant conformational changes of the residues that actually interact with the drugs. While this is an intriguing result, the authors should look into (using in-silico strategies)/comment on how these mutations can potentially modulate corresponding host-viral interactions (of nsp5 and nsp12).

2. Do the observed resistance mutations occur at interfaces of host-viral interactions? This can be investigated using publicly available resources (e.g., https://www.nature.com/articles/s41592-021-01318-w) and will provide additional insights into the potential immunomodulatory effects of these mutations.

3. While the in-vitro antiviral binding assays used in the study are reasonable, they have limitations and provide relatively crude estimates of actual antiviral neutralization. The authors should better discuss this.