Functional and Evolutionary Characterization of the NSP6 Protein in SARS-CoV-2 Omicron Variants
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsLine 76: RdRp: can you explain the meaning?
Line 76: remdesivir: can you classify this medication?
Line 95: formed from the ER: can you explain this expression?
Line 106: further research is: or researches are .....?
The introduction and the results are well described. I think it is the case to improve the discussion and the results, in way to highlight what is the contribution of your paper to the research.
In particular, can you predict the more probable NSP6 evolution in South America?
Can you outiline the NSP6 characterization in South America compared to USA and Europe?
Author Response
Comments 01 - Line 76: RdRp: can you explain the meaning?
R: To improve the text we have inserted the following information "The RdRp (RNA-Dependent RNA Polymerase) is a key NSP responsible for the synthesis of viral RNA during replication, the RNA-synthesizing machinery in coronaviruses requires incorporation of RdRP together with other key nsps to form a fully functional polymerase complex"
Comments 02 -Line 76: remdesivir: can you classify this medication?
R: "including remdesivir, It was initially developed to treat hepatitis C, later explored for its potential against Ebola virus disease and Marburg virus infections, and eventually studied as a treatment for COVID‑19 after infection."
Comments 03 -Line 95: formed from the ER: can you explain this expression?
R: A NSP6 protein can be found in the endoplasmic reticulum (ER) and Golgi apparatus of infected cells com SARS-CoV-2 and has two transmembrane domains anchoring it to the ER membrane, with N- and C-terminal domains located in the cytosol.
Comments 04 - Line 106: further research is: or researches are .....?
R: The text has been changed for better understanding
Comments 05 -The introduction and the results are well described. I think it is the case to improve the discussion and the results, in way to highlight what is the contribution of your paper to the research.
R: "In the context of the COVID-19 vaccine, the NSP6 protein plays an important role in regulating the immune response and modulating cellular functions. Changes in the hydrophobicity and hydrofisibility of NSP6 may impact its ability to interact with cell membranes and, consequently, influence the efficacy of vaccines that use viral proteins as a base. Therefore, it is essential to understand how the biochemical characteristics of this protein evolve, especially in critical regions such as those highlighted in the graphs.
Mutations observed in NSP6, such as those in the regions between positions 100–150 and position 250, may alter its functionality and pathogenic potential. Alterations that increase hydrophobicity may favor its association with cell membranes, impacting its ability to form transmembrane structures and interfering with intracellular trafficking, a mechanism often associated with immune evasion. These changes may require adjustments in vaccine formulation to ensure that immune responses are adequate to emerging variants."
Comments 06 -In particular, can you predict the more probable NSP6 evolution in South America?
R: In our study, although we performed phylogeny, we did not focus on the evolutionary process and ancestry of the samples. Instead, we only aimed to demonstrate that they have specific mutations. To do this, we would have to modify our approach, which we believe is not interesting at this time.
Comments 07 -Can you outiline the NSP6 characterization in South America compared to USA and Europe?
R: we have added new information about the characterization of the strains used
Reviewer 2 Report
Comments and Suggestions for AuthorsThis is an interesting paper but there are some serious shortcomings.
1) The resolution of Figure 3 is so bad that it is totally useless. I am unable to see a single thing even if I try to enlarge it. Part of the reason is that the authors are trying to put too much information into the tree. That is fine except even if the reader is able to read it, it will be hard to read because there is just too much information. It is alright if you have a tree like this as one of the images but there should be another much smaller tree that is more easily readable with only selected more important viruses used.
2) The discussions and results in this paper are simply too inadequate. There is much to discuss but it is not. For example, the paper says there are three groups in the phylogenetic tree but it fails to discuss further or give more description of the three groups. What are the viruses in each group? What are the differences? How close are they to previous variants?
3) No discussion on why they are virulent or not virulent. Infectivity.
4) Where are the mutations? What are they doing in terms of binding? Where is it binding? Where it is not? Is there literature on it?
5) I am not sure what they have shown or what is the significance of their results. They must show this and they have not shown this.
Author Response
1) The resolution of Figure 3 is so bad that it is totally useless. I am unable to see a single thing even if I try to enlarge it. Part of the reason is that the authors are trying to put too much information into the tree. That is fine except even if the reader is able to read it, it will be hard to read because there is just too much information. It is alright if you have a tree like this as one of the images but there should be another much smaller tree that is more easily readable with only selected more important viruses used.
R: We appreciate your consideration and have verified that the image quality needed adjustment, so we created a new image to make it more interesting.
2) The discussions and results in this paper are simply too inadequate. There is much to discuss but it is not. For example, the paper says there are three groups in the phylogenetic tree but it fails to discuss further or give more description of the three groups. What are the viruses in each group? What are the differences? How close are they to previous variants?
R: Thank you for your considerations, based on the questions, include more information and a better description of the samples.
3) No discussion on why they are virulent or not virulent. Infectivity.
R: Although our focus is not on whether or not the virulence of the virus increases, but rather on the changes that may alter recognition in relation to vaccine action, we have included in the text a brief discussion on NSP6 and its relationship with the increased virulence of SARS-CoV-2.
4) Where are the mutations? What are they doing in terms of binding? Where is it binding? Where it is not? Is there literature on it?
R: We have provided a new description of the mutations, making them clearer, for example, in Table 01. We have also included information about NSP6 and the virulence of SARS-CoV-2 based on our findings and what is described in the literature.
5) I am not sure what they have shown or what is the significance of their results. They must show this and they have not shown this.
R: We believe that the changes suggested in our manuscript make our real intention clearer and we are aware that our data can help in better understanding NSP6 and its functions and relationships mainly with the transmission of SARS-CoV-2.
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors1)I don't agree with the sentence on line 31. It is wrong. The envelope is NOT "derived" from S..etc. It includes or is made up of of S...etc.
2) It is very awkward to have a paragraph with 1-2 sentences such as line 54.
3)Figure 5 is still impossible to read even when you try to enlarge the paper. I would advise the figure be broken into 4 separate parts. The main part containing only the 3 main branch while each of the 3 other parts containing one of the 3 main branches. Please make sure that the figure(s) presented is readable. Otherwise, the paper is simply not publishable.
4) Flexibility or disorder in a protein may mean that the protein can recognise tis binding partners more efficiently:
https://pubmed.ncbi.nlm.nih.gov/10550212/
Author Response
1)I don't agree with the sentence on line 31. It is wrong. The envelope is NOT "derived" from S..etc. It includes or is made up of of S...etc.
We corrected the sentence
2) It is very awkward to have a paragraph with 1-2 sentences such as line 54.
We corrected the sentence
3)Figure 5 is still impossible to read even when you try to enlarge the paper. I would advise the figure be broken into 4 separate parts. The main part containing only the 3 main branch while each of the 3 other parts containing one of the 3 main branches. Please make sure that the figure(s) presented is readable. Otherwise, the paper is simply not publishable.
We appreciate the information and have decided to remove the figure.
4) Flexibility or disorder in a protein may mean that the protein can recognise tis binding partners more efficiently:
https://pubmed.ncbi.nlm.nih.gov/10550212/
We have inserted new information
Round 3
Reviewer 2 Report
Comments and Suggestions for AuthorsFigure 3 is missing even though the legend is there. There should also be further descriptions of the figure in the legend
Author Response
Dear all,
We appreciate the opportunity and would like to point out that we have adjusted the request:
The caption that mentions figure 03, which no longer exists in the manuscript, has been removed.
Author Response File: 
Author Response.docx
Round 4
Reviewer 2 Report
Comments and Suggestions for AuthorsImprovements seen.
Author Response
We appreciate the opportunity and have corrected the requested subtitles.
