Identifying Viral Protein Interactions’ Order During Replication and Transcription Processes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

There are major concerns regarding the overall description. The units of physicochemical parameters related to the values ​​described are not stated in the text or in the figures and tables. This is essential for biophysics papers. Unless these are added, the paper will not be accepted as a scientific paper. Biophysical researchers actually pay great attention to this point when writing biophysics papers. cal/mol (calories per mole) has historically been used in biophysics papers, but J/mol (joules per mole), which is an SI unit, is now recommended. Therefore, the lack of units is a major flaw, and adding them will be the subject of review. Even if it is stated in the text that the unit system can be omitted, this is not generally accepted.There is no way to comment unless these are stated.

Author Response

Thank you for your insightful comments regarding the importance of specifying
units in biophysical research. We have now revised the manuscript to ensure that
all physicochemical parameters are clearly labeled with their respective units in the text,
figures, and tables. For example, the dissociation constant Kd is explicitly stated in molarity
(M, mol/L), and when logarithms are taken, they refer to dimensionless quantities.
We have also adopted units throughout the paper, using kcal/mol where applicable, to
meet the current standards in the field. We trust that these revisions fully address your
concerns regarding unit specifications.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper "Identify viral protein interactions order during replication and transcription processes" focuses on the thermodynamic parameters involved in the assembly and stability of molecular complexes, particularly in viral replication processes. The authors analyze biochemical pathways through experimental and theoretical approaches, exploring how mutations impact complex stability and binding affinity across various protein and RNA interactions. The manuscript presents a comprehensive methodology, combining experimental data with computational models to offer insights into molecular assemblies and their thermodynamic landscapes. The publication is fit to be accepted after considering minor revisions.

1. The manuscript presents detailed thermodynamic parameter calculations for different LGP2 mutants, such as I597S and K634E. However, it remains unclear how the observed trends in stability and entropy (TΔS) specifically correlate with the biological function of these mutations. Could the authors elaborate on the biological significance of these mutations in the context of the stability of intermediate complexes compared to the final [LGP2-8dsRNA-LGP2] assembly? This explanation would help clarify whether increased stability is advantageous for biological function.
2. In Figure 4d, the transition from negative to positive dissociation energy values for intermediate complexes is intriguing. Could this shift represent an energy barrier that prevents non-specific binding or an essential step for ensuring correct assembly? The authors are encouraged to discuss whether this transition reflects a regulatory mechanism or a kinetic checkpoint during the forming of the final molecular complex.
3. The manuscript describes how specific mutations in VP35 (R305A, K339A) affect the electrostatic surface of the protein-RNA interface. While the effect on binding affinity is noted, it would be valuable to expand on how these electrostatic changes influence the biological function of VP35 in immune evasion. Furthermore, could these insights be used to propose new therapeutic strategies for targeting Ebola virus replication? A brief discussion in the concluding section would strengthen the paper's impact.
4. The manuscript provides an excellent description of the effect of mutations such as R305A and K339A on the electrostatic surface of the VP35-dsRNA interaction. However, it remains unclear how these surface changes translate to functional outcomes at the molecular or biological level. Could the authors clarify whether the observed shifts in lg[Kd] and TΔS values directly impact viral replication efficiency or immune evasion mechanisms? This information would strengthen the biological relevance of the study.
5. In Figure 7, significant variations in lg[Kd] values are reported for intermediate complexes during the tetramer formation process. While the data suggest confident intermediates are less stable, the authors should discuss how these intermediates represent kinetic bottlenecks or potential regulatory checkpoints in complex assembly. Are these intermediates biologically relevant, or could they be artifacts of the modeling approach?
6. The transition from high to low lg[Kd] values during tetramer assembly is well described. However, the manuscript does not fully explore the potential energy landscape associated with these transitions. Could the authors elaborate on whether the observed patterns in lg[Kd] correspond to distinct energy minima and whether these represent preferred assembly pathways or metastable states? This would provide deeper insights into the thermodynamic landscape.
7. The R322A and H240A mutations significantly alter the assembly pathway by shifting the complex toward higher entropy and lg[Kd] values. Could the authors discuss whether these mutations - or similar surface alterations - could serve as targets for therapeutic interventions? Additionally, does this finding suggest a broader strategy for disrupting viral protein assemblies in related systems?
8. The manuscript highlights the presence of dead-end formations such as [P1 Pep2 P3 P5] 3.3 with low lg (cond(W)) but high lg (Kd) values. Could the authors clarify whether these dead-end complexes represent biologically relevant intermediates or modeling artifacts? Additionally, how do these dead-end states impact hexamer formation's overall efficiency and likelihood under physiological conditions?
9. Figure 14 shows the correlation between experimental Kd values and calculated thermodynamic parameters such as lg (Kd), T∆S, and ∆(∆W). However, some data points deviate from the expected trend. Could the authors discuss potential reasons for these discrepancies? Are these deviations attributable to computational model limitations, experimental measurement inaccuracies, or inherent variability in peptide-protein interactions?

Author Response

We tried to answer all questions

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript biophysica-3470416 is very confused and must be rewrite to be evaluated again.

1) At least triplicate assays must be done to verify the trend. Please, do it and insert the corresponding standard deviation in the plots.

2) The way that the manuscript was written is very confusing. It does not sound like an article but like a Review. Please, restructure the manuscript following the guidance of MDPI.

3) The authors showed ITC data, however, they did not provide calorimetric titration. Please, provide it as supplementary material.

4) What were the concentrations and conditions used in the ITC assays? Provide it.

Author Response

We tried to answer all questions

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors of the manuscript biophysica-3470416 replied to the Reviewer’s comments and improved the quality and comprehension of the work. Thus, I recommend the publication.