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AK acknowledges the financial support from NIH Grants R01GM127701 and R01HG012117. MH acknowledges the Ohio State University for providing the "President’s Postdoctoral Scholars Program (PPSP)” award for financial support to complete this computational research.

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Additional Editor Comments :

While the source of primary datasets and tools is declared, stronger metadata reporting is suggested. For example, provide accession numbers, precise parameter sets, and, if possible, deposit final structure files and input scripts in a persistent public repository (such as Zenodo or Figshare) for full reproducibility.

While the manuscript’s focus is on RSV, a paragraph, possibly in the Discussion, could briefly contrast host miRNA targeting of RSV with its role in related viruses (such as Influenza, Metapneumovirus, or Parainfluenza), identifying any common antiviral targets or motifs.

The manuscript uses miRanda and RNAhybrid, but does not comment on why these were prioritized over newer or ensemble prediction platforms. A concise justification or reference to benchmark comparisons could contextualize the strengths of the chosen tools and identify best practices for future studies.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: It was a privilege to review your manuscript entitled, “Assessment of miRNAs as Transcriptional Regulators in Respiratory Syncytial Virus Infection through Bioinformatics Analysis and Molecular Docking Studies.” I commend the authors for their effort in conducting this work. The authors put in a lot of thoughtful and thorough consideration into the work. The topic is timely and has the potential to be impactful. The manuscript is well-structured. I appreciate the clarity of the research question and the contribution it seeks to make to the existing body of knowledge. I appreciate the opportunity to provide some constructive feedback that may help strengthen the manuscript and improve its clarity and methodology for the global readership.

Overall assessment: The topic is timely, and the multi-layered in-silico pipeline is a reasonable discovery framework.

Strengths

• Clear biological motivation linking host miRNAs to viral replication control.

• The methodology is impressive and thorough. Its consistent findings identify hsa-miR-2278 and hsa-miR-6732-3p for further study in future research, both clinical and academic.

• Cross-tool nomination using both miRanda and RNAhybrid yields a consensus set (n=12), a sensible first filter.

• Attempts at structural plausibility (miRNA-mRNA docking) and basic stability checks using MD simulation analysis demonstrate a multi-layered in-silico pipeline.

• The discussion touches on translational pathways (e.g., nanoparticle delivery), which can be developed further.

However, there are substantial issues with dataset/version transparency, target-prediction controls, orientation/accessibility, docking/MD rigor, grammar and sentence composition, and reporting/reproducibility. With focused revisions and clearer statistics, this work could be suitable after a major revision. I hope my input as a peer reviewer will help to make this paper stronger and ready for the target audience.

Section-by-section comments

Title & Abstract

• Abstract should report numbers (48 vs 312 predictions; 12 overlaps) and the criteria used.

• The RNAhybrid should be spelled correctly and consistently throughout the paper (not RNAHybrid) to preserve the credibility of the work.

Introduction

Nice overview of RSV proteins and miRNA biology. Please:

• Fix typos: “headacheg” → headache; “perfusion protein F1” → prefusion protein F.

• Report the RSV taxonomy choice consistently and accurately. The Introduction cites Pneumoviridae (correct for RSV), but the author later refers RSV to the Paramyxoviridae family further in Section 2.6. Resolve this taxonomic inconsistency.

• “MicroRNAs (miRNAs) are non-coding RNAs control the transcription/translation profiling based on sequence complementarity.” → MicroRNAs (miRNAs) are non-coding RNAs [that] control the transcription/translation profiling based on sequence complementarity.

o This is one of several sentences where the idea that the authors intend to convey is lost due to typographical errors or an inaccurate grammar construct. I will recommend that the authors employ the services of a professional writer or scientific proofreader to assist with improving the writing.

• “These protein plays…”

o For subject-verb agreement, the sentence should read, “These proteins play…”. Subject-verb agreement errors occurred in several parts of the manuscript. Correcting these errors will improve this paper for the target readers.

Methods vs Results and Discussions:

While the authors opted to go straight to Results and Discussions after the Introduction, it is my opinion that the manuscript flowed better with the Methodology section coming before the Results and Discussion section; it provides a good lead-in that makes it easier to process the results discussed following.

Methods (Computational)

The computational Methodology is detailed and well-articulated, including algorithm parameters, tool/database versioning, and reference citations.

3.2 RSV genome retrieval

• The authors retrieved “360 human miRNAs” from miRBase. Contemporary human miRNA catalogs contain far more entries; if the restriction to 360 miRNAs was intentional, the methodology should include your inclusion criteria or restraints.

• Provide the exact miRBase release number and date.

3.3 Target prediction

• Report exact miRanda (version; score/energy thresholds, penalties) and

• As mentioned above, ensure that RNAhybrid is correctly spelled. RNAhybrid settings and the rationale.

Results

2.1 RSV Genome organization

• This section was well written and provides the background for the discussion of the result that was to follow. Viral protein mapping provides a good visualization.

• Ensure taxonomy is correct and consistent.

• Formatting Tables and Figures labels - should be distinctly formatted from the general text of the manuscript to make it easier for the readers to identify and assign to the image, figure, or table that they label. Examples will be to indent or make the label text of a different font size and/or justification.

2.2 Target predictions

• 2.2.1: Statistical control and enrichment - Show that the 12 overlapping miRNAs (miRanda ∩ RNAhybrid) exceed chance expectation. Provide:

o A null model (e.g., shuffled miRNAs or shuffled target segments) to estimate empirical FDR.

o Full score/MFE distributions and selection cutoff

• In literature, the first mention of an abbreviation should be written out and consequently abbreviated. The mention of MFE in subsection 2.2.2 should be preceded by the full words written out.

• Pay attention to inconsistent use of the word “grey” vs “gray.” It is important to be consistent. “Grey – with an e” is preferred for a global audience. Review the article for the words and adjust as appropriate.

• Clarify sign conventions (e.g., “G protein exhibited highest free energy” - do you mean least negative MFE, i.e., less favorable? Be precise).

2.3 Protein sections

• 2.3.3 - Correct the typo: prefusion protein, F1 (not perfusion protein)

Pay attention to punctuation throughout this section, as some of the sentences lack clarity owing to improper punctuation. For example, “The M2 gene encodes two proteins[,] M2-1 and M2-2.

• 2.3.7 – “The features of G and its ability to block its function with antibodies…”

o This sentence is unclear.

2.4 mRNA-miRNA docking analysis.

• Good qualitative description of hydrogen bonds (H-bonds).

• Avoid over-interpreting single bond distances; aggregate metrics are more meaningful.

• 2.4.2.1 – “In miR-200c docking couple of hydrogen bonds have been observed with different nucleotides at different positions.”

o It is unclear to me what the author is attempting to convey. Between sentence construction and possible missing words.

• Standardize angstrom notation (Å) and keep the spacing between the digits and the unit (Å) consistent throughout the manuscript – 1,45 Å or 1.45Å.

• There are several typographical errors and sentence constructions to fix in this section of the manuscript, for which I recommend the use of a professional writer to assist with.

o cytocine” → cytosine, “bind formation” → bond formation; “nonapplicable” → not applicable.

• 2.4.2.2 – “Furthermore, cytosine and adenine at positions 6 and 7 forms hydrogen bonds with mRNA nucleotide C-36 and uracil 48 (U-48) with bond distances 2.485 Å and 2.166 Å, respectively.”

o Review for better clarity and eliminate inconsistencies noted with nucleotide bases and positions throughout this section - “cytosine at position 6 (C-6), Uracil 48 (U-48), C-36.

• Use standardized names (e.g., hsa-miR-200c-5p, hsa-miR-204-3p) consistently. Several instances mix “has_,” “miRNA_,” and missing arm designations.

2.5 MD (RMSD/RMSF/SASA/Rg)

• Write abbreviations’ full words out at first mention – RMSD, RMSF.

2.6 miRNAs as transcriptional profilers in RSV infection

• “RSV is a non-segmented negative sense RNA virus belonging to the Paramyxoviridae family and is responsible for significant numbers of severe human infections each year.”

o Ensure to correct the Taxonomy of RSV to “Pneumoviridae.”

Other General Comments:

• Spelling/typos: “headacheg” → headache; “cytocine” → cytosine; “perfusion protein F1” → prefusion F; “bind formation” → bond formation; “nonapplicable” → not applicable.

Recommendations

Thank you for the opportunity to peer review this manuscript. The biological rationale is strong, and the pipeline is sensible. The manuscript needs clearer data/versioning, statistical controls, in addition to some major revisions in writing. Addressing these items that I have highlighted above will materially strengthen the paper and its translational credibility.

Reviewer #2: Dear Editor:

Thanks for the opportunity reviewing this paper, here is my comments related o this work:

The manuscript addresses a valuable and relevant topic to the field of host-virus interactions, specifically focusing on miRNA-mediated regulation in RSV infection. The authors have employed a sophisticated and comprehensive computational biology pipelines, including: accessing of multiple databases to collect miRNA and mRNA, 3D modeling, visualization, molecular docking, and molecular dynamic simulations. The introduction is written well, abstractive and informative and the specific knowledge gap is clear.

However, significant revisions are required to bring the manuscript up to the publishing standards of a Q1 journal. The major concerns are structural, contextual, and foundational regarding the input data.

1. Manuscript structure:

The author need to follow journal guidelines for manuscript structure for example discussuin and result should be separated.

2. Graphical abstract:

The multi-step, sophisticated pipeline is complex and maybe dificault for readers to follow in text alone. Therefore, addition of a graphical abstract is highly recommended. This visual should clearly outline the entire workflow, from the initial number of miRNAs considered to final 2 miRNAs suggested target.

3. Methodology

3.2 Retrieval of Mature miRNAs of Humans

Clarification of initial miRNA Dataset:

1. The author states that “360 human miRNAs …. Were retrieved from miRBase”. This implies that the total set of human miRNAs in the used version of miRBase is 360. The mentioned number maybe consistent with a very outdated release of the database. The current miRBase contains around 2,000 human miRNAs.

Please specify the exact version of miRBase used and justify the use use of sich an outdated dataset, or preferably, repeat the analysis with the current version.

2. The author should clarify that: does the number 360 refer to:

a. the total number of human miRNAs downloaded from miRBase before running any further prediction?

OR

b. The final number of miRBase predicted by miRanda/RNAHybrid to target RSV?

If it is (a), the version number is critical. If it is (b), this result belongs in the results section, and the methods must describe the complete set of miRNAs used as input for the prediction tools.

4. Discussion

The discussion appears to lack support from relevant scientific literature. The findings are not adequately contrasted with or supported by prior published studies. The discussion section should be further strengthen by integrating references to similar computational or experimental studies on miRNA-virus interactions for RSV or related viruses like Influenza.

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Reviewer #1: Yes: Babajide AdewumiBabajide Adewumi

Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.-->

Assessment of miRs as Transcriptional Regulators in Respiratory Syncytial Virus Infection through Computational Analysis and Molecular Docking Studies

PONE-D-25-38737R1

Dear Dr. Hassan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Abayeneh Girma

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: (No Response)

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3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: (No Response)

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: (No Response)

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: (No Response)

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Reviewer #1: he authors have adequately addressed all reviewer and editorial comments. The revised manuscript complies with the journal’s submission guidelines, and no further revisions are required.

Reviewer #2: (No Response)

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what does this mean? ). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy .-->

Reviewer #1: Yes: Babajide AdewumiBabajide Adewumi

Reviewer #2: Yes: ALI ABDULQADERALI ABDULQADER

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