Dear Dr. Smit,

Thank you very much for submitting your manuscript "Chikungunya virus requires an intact microtubule network for efficient viral genome delivery" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Abdallah Samy

Deputy Editor

PLOS Neglected Tropical Diseases

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Yes to most questions, other than what's included in my comments below.

No ethical/regulatory concerns.

Reviewer #2: Appropriate. It would have been useful to include an indication of the particle to infectious unit ratio, especially for the DiD labelled virus.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Please see specific comments below

Reviewer #2: The results are for the most part clearly presented. I have just a few questions/comments.

Firstly, the authors show that cell viability is unaffected after 18hrs of nocodazole treatment (Fig S1B), but presumably cell division is inhibited and there are fewer cells in these cultures? If this is the case, does it impact on the data presented in S2B - if there are fewer cells in the nocodazole treated samples, presenting the data as the percentage of infected cells will underestimate the nocodazole inhibition? What are the dotted lines in S1B and S3B?

Why have authors chosen to assess the effects of nocodazole at 10 hrs post infection?

Using DiD as a fusion reporter does detect hemifusion, but it also reports full fusion. In these assays the authors cannot distinguish between hemifusion and full fusion. So I think it misleading to use hemifusion throughout the paper, and I suggest they use 'hemifusion/full fusion' instead.

Can the authors be certain that the fast directed movement described in Fig 2A is intracellular and not on the cell surface? I.e. could it be viral surfing?

Line 291 - there does seem to be a 30% change in MFI.

Figs 2A and B - the labeling of the boxes is defective.

Line 375 - how do the authors define perinuclear? The two events illustrated in Fig 2A do not appear to be perinuclear, yet in Fig 3/Line 398 the authors give a quantitative analysis of perinuclear v non-perinuclear fusion.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Yes to all ?s.

Reviewer #2: The conclusions are appropriate for the data presented. However, I am concerned that the authors do not cite and discuss relevant published work appropriately. Specifically, papers by Vonderheit and Helenius (PLoS Biol 2005 e233) describing morphological analysis of Semliki Forest virus is not mentioned; and though cited, Bernard et al's. report of clathrin-independent internalization of CHIKV into cells is not discussed.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Please see specific comments below

Reviewer #2: The paper should be carefully edited for word usage, etc.

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: In this manuscript, Hoornweg et al elucidate the role of the microtubule network in the trafficking of CHIKV particles early in the course of infection. They hypothesize that microtubules may direct the particle to a cellular location that is beneficial for establishing infection or aids in nucleocapsid uncoating. They monitored trafficking of DiD-labelled viral particles and observed two distinct patterns prior to membrane fusion in the early endosomes. Using nocodazole to disrupt the microtubule network, they observed reduced number of infected cells, restriction of fusion to the cell periphery, and impaired delivery of the viral genome into the cytoplasm.

The microtubule network has been previously implicated in the entry of multiple other viruses, and thus the innovation here is limited to the demonstration of this requirement for CHIKV and the discovery of the two distinct trafficking patterns. While the data demonstrating the two distinct viral populations is interesting, it remains unclear what is the functional relevance of this difference and what is the mechanistic role mediated by microtubules in CHIKV entry.

Major comments:

1. The magnitude of the effect shown in most figures is small (less than a 2 fold difference in many cases – e.g. Fig. 1A, Fig. 4B), which makes it unclear what the biological relevance of the findings is.

2. The number of infected cells is very low (2.9% in an MOI of 1, 9.2% in an MOI of 20 as per line 288). Why did the authors choose to infect the cells only for 30 min (when a more standard infection time is 1 hour for alphaviruses)? Perhaps a longer infection time will increase infection rate and improve the dynamic range?

3. The authors should demonstrate a dose-response effect for the various phenotypes shown with nocodazole treatment.

4. Is it possible the cell cycle arrest at G2/M induced by nocodazole accounts for the observed reduction in infection and trafficking of viral particles?

5. The distance and velocity traveled by individual viral particles needs to be shown in figures 2 and 3.

6. In Fig. 4A, the Western blot should include all 3 conditions (control, and two treatments).

Additional comments:

1. Graphs in all figures – would be better to show controls in each figure rather than showing just the % (or alike) difference relative to control.

2. Would be good to avoid single column graphs (as in 1D). These can be just mention in the text.

3. Figure 1E – a schematic showing time of drug addition would be helpful.

4. Figure 3D – the legend should explain what before, during and after means.

5. Embedding figure legends in the text makes it harder to review.

Reviewer #2: Overall, this work provides a modest step forward in understanding CHIKV entry into cells. Unfortunately, there is really no indication of how microtubule based transport of CHIKV favors penetration and infection.

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Reviewer #2: No

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Dear Dr. Smit,

We are pleased to inform you that your manuscript 'Chikungunya virus requires an intact microtubule network for efficient viral genome delivery' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Abdallah M. Samy, PhD

Deputy Editor

PLOS Neglected Tropical Diseases