Modulation of Biophysical Properties of Nucleocapsid Protein in the Mutant Spectrum of SARS-CoV-2

Reviewer #1 (Public Review):

The study is highly interesting and the applied methods are target-oriented. The biophysical characterization of viable N-protein species and several representative N-protein mutants is supported by the data, including polarity, hydrophobicity, thermodynamic stability, CD spectra, particle size, and especially protein self-association. The physicochemical parameters for viable N-protein and related coronavirus are described for comparison in detail. However, the conclusion becomes less convincing that the interaction of peptides or motifs was judged by different biophysical results, with no more direct data about peptide interaction. Additionally, the manuscript could benefit from more results involving peptide interaction to support the author's opinions or make expression more accurate when concerning the interaction of motifs. Although the authors put a lot of effort into the study, there are still some questions to answer.

Reviewer #2 (Public Review):

Summary:
This work focuses on the biochemical features of the SARS-CoV-2 Nucleocapsid (N) protein, which condenses the large viral RNA genome inside the virus and also plays other roles in the infected cell. The N protein of SARS-CoV-2 and other coronaviruses is known to contain two globular RNA-binding domains, the NTD and CTD, flanked by disordered regions. The central disordered linker is particularly well understood: it contains a long SR-rich region that is extensively phosphorylated in infected cells, followed by a leucine-rich helical segment that was shown previously by these authors to promote N protein oligomerization.

In the current work, the authors analyze 5 million viral sequence variants to assess the conservation of specific amino acids and general sequence features in the major regions of the N protein. This analysis shows that disordered regions are particularly variable but that the general hydrophobic and charge character of these regions are conserved, particularly in the SR and leucine-rich regions of the central linker. The authors then construct a series of N proteins bearing the most prevalent mutations seen in the Delta and Omicron variants, and they subject these mutant proteins to a comprehensive array of biophysical analyses (temperature sensitivity, circular dichroism, oligomerization, RNA binding, and phase separation).

Strengths:
The results include a number of novel findings that are worthy of further exploration. Most notable are the analyses of the previously unstudied P31L mutation of the Omicron variant. The authors use ColabFold and sedimentation analysis to suggest that this mutation promotes the self-association of the disordered N-terminal region and stimulates the formation of N protein condensates. Although the affinity of this interaction is low, it seems likely that this mutation enhances viral fitness by promoting N-terminal interactions. The work also addresses the impact of another unstudied mutation, D63G, that is located on the surface of the globular NTD and has no significant effect on the properties analyzed here, raising interesting questions about how this mutation enhances viral fitness. Finally, the paper ends with studies showing that another common mutant, R203K/G204R, disrupts phase separation and might thereby alter N protein function in a way that enhances viral fitness.

Weaknesses:
In general, the results in the paper confirm previous ideas about the role of N protein regions. The key novelty of the paper lies in the identification of point mutations, notably P13L, that suggest previously unsuspected functions of the N-terminal disordered region in protein oligomerization. The paper would benefit from further exploration of these possibilities.

Reviewer #3 (Public Review):

Nguyen, Zhao, et al. used bioinformatic analysis of mutational variants of SARS-CoV-2 Nucleocapsid (N) protein from the large genomic database of SARS-CoV-2 sequences to identify domains and regions of N where mutations are more highly represented and computationally determined the effects of these mutations on the physicochemical properties of the protein. They found that the intrinsically disordered regions (IDRs) of N protein are more highly mutated than structured regions and that these mutations can lead to higher variability in the physical properties of these domains. These computational predictions are compared to in vitro biophysical experiments to assess the effects of identified mutations on the thermodynamic stability, oligomeric state, particle formation, and liquid-liquid phase separation of a few exemplary mutants.

The paper is well-written and easy to follow, and the conclusions drawn are supported by the evidence presented. The analyses and conclusions are interesting and will be of value to virologists, cell biologists, and biophysicists studying SARS-CoV-2 function and assembly. It would be nice if some further extrapolation or comments could be made regarding the effects of the observed mutations on the in vivo behavior and properties of the virus, but I appreciate that this is much higher-order than could be addressed with the approaches employed here.

Author Response

We thank the editors and reviewers for taking the time to provide a critical assessment of our manuscript. We are delighted our work was found to have merit, and will revise the manuscript based on their valuable input.