Inhibition of Rho Kinase or Myosin-light chain kinase decreases Dengue virus replication in endothelial cells

  1. Gena Nichols
  2. Andrea Spengler
  3. Thomas G. Voss

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Abstract

Dengue virus (DV), a member of the Flaviviridae family, has been demonstrated to induce changes to the actin cytoskeleton to promote efficient viral entry and replication. These cytoskeletal alterations continue throughout infection, and disruption of the cytoskeleton has been shown to decrease the release of infectious virions. However, few studies have assessed the contribution of actin alterations to Dengue-induced vascular leak in endothelial cells. A number of cellular kinases control the assembly and rearrangement of actin filaments, including Rho-activated kinase (ROK) and Myosin Light Chain kinase (MLCK). To determine whether the observed changes to the actin cytoskeleton during Dengue viral infection contribute to the loss of endothelial cell barrier function, the roles of the cellular kinases ROK and MLCK were evaluated during DV infection. While kinase inhibition did not prevent virus-induced changes in monolayer permeability, specific inhibition of either kinase resulted in a significant decrease in virus yield indicating that ROK/MLCK-mediated cytoskeletal alterations play a role in virus replication and biogenesis that do not affect vascular integrity in Dengue virus infection in vitro.  Further investigation revealed that ROK or MLCK inhibition decreased virus yield, but only MLCK inhibition decreased viral protein synthesis and genome replication. Taken together, these results identify a role for both ROK and MLCK activity in the efficient replication of Dengue virus in endothelial cells, potentially affecting the assembly or budding of mature Dengue virions.

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  2. Version published to 10.1099/acmi.0.000845.v2 on Access Microbiology
  3. Access Microbiology

    the reviewers were largely positive about this manuscript, but have expressed some concerns about the conclusions drawn based on the data. In your revision, please address all reviewer comments and pay particular attention to supporting any claims and conclusion with your data.

  4. Access Microbiology

    Comments to Author

    The paper introduces a mechanism by which kinases (ROK and MLCK), that contribute to actin alterations, play a role in the the Dengue virus replication in endothelial cells. It shows how inhibition of the two kinases affect the permeability of Dengue virus-infected endothelial cells, virus yield, and viral protein and RNA expression. The methodology that the authors used were appropriate for the hypotheses tested. The use of TEER and HRP flux assays to measure endothelial cell permeability gave reliability to the results. The presentation of results using fluorescence micrographs and several bar graphs presented the results in simple manner that is easily understandable. The way the paper is written follows a clear narrative of what the authors wanted to investigate, providing proper explanation and rationale for every method, clear presentation of results, and provision of well thought-out discussion. Key findings were presented in a straight-forward manner. However, I have some clarifications to make in terms of interpretation of results: Lines 233-235: You only addressed the results at 24, 48, and 72 hpi timepoints. How about the result at 96 hpi, which seems to show a relatively higher HRP? Lines 267-268: I don't see a decrease in the yield of non-treated cells at 72 hpi based on Figure 3A. Can you clarify this in the context of comparing the results for treated cells? The Y27632 treatment did not seem to affect the number of progeny from 48 to 72 hpi while the ML7 treatment did not seem to stop the increase in progeny from 48 to 72 hip as seen in the increase. Lines 288-291: How can you discount the fact that the observed result was due to the cytotoxic side effects of the addition of the inhibitors? A few very minor revisions are suggested for the manuscriipt. They mostly include typographical errors, consistency issues, and units. Kindly revisit the following and revise as necessary: Line 87: should the word "import" be revised to "importance"? Line 139: I assume that "Immunofluorescence staining" is a subheading and therefore should be a new line and should have the bold text. Line 141: The acronym presented here for multiplicity of infection is "MOI" (all caps). However, the rest of the manuscript uses "moi" (small letters). I suggest revising for consistency. Lines 191-192: Please indicate the unit for temperature "C"

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  5. Access Microbiology

    Comments to Author

    This is a well presented and written paper adding important data to our understanding of cell biology during a Dengue infection. However there are a number of issues that should be addressed before publication. General: There are a number of typos/formatting issues to be corrected i.e. Line 85: double full stop Line 139: Immunofluorescence staining. Looks like it was meant to be a section title The manuscript will require some further proofreading and reformatting before final submission. Singh et. al. (DOI: 10.1099/jgv.0.001163) demonstrated that the NS1 protein from Dengue was key in cell permeability during infection. The authors may not have looked at this element but it would be useful to include some reference to this work to help put this manuscript into a broader context. Methods: Line 191: Temperatures are presumably in Celsius but this should be indicated. Figures: In general require reformatting (Fig 3 and 5 are not connected to their captions) Fig 4 appears to be missing and the reference to figure 4 B (line 317) appears to refer to what is currently Fig 5 C. This will need to be corrected and figures reformatted and repositioned as they currently partially cover text. Fig 5 (4?): section A seems to indicate that E protein is expressed, although certainly reduced in the presence of ML7, however section C suggests that there is no increase in gene copy numbers at all is this connected to sensitivity of the RT?

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  6. Access Microbiology

    Comments to Author

    Thank you for this submission and congratulations on completing the study. I found the manuscript easy to read in general and the experiments were well explained. I particularly enjoyed the detail in the materials and methods. Including information such as antibody dilutions is rare but appreciated! Unfortunately, I did have some difficulties with this manuscript. My major criticism is that a lot of the discussion and conclusions are not supported by the results presented. Here are some particular points for your consideration: The title contains abbreviations that are not explained (ROK, MLCK) - try to avoid this. The title says that Dengue virus replication is restricted by inhibitors of ROK or MLCK. I don't think the data presented supports that statement so putting it in the title is questionable. In Line 27 you state that ROK and MLCK prevent stress fiber formation. This is reiterated on line 106. In the results section, Line 218, this is described as ROK or MLCK causing 'somewhat reduced stress fiber formation'. Finally in the discussion, Lines 337-339' you state 'In this report, we demonstrate that Dengue infection induces actin stress fiber formation but that ROK and MLCK signaling pathways do not seem to be the major effectors of this change, as inhibition of either kinase only minimally prevented stress fiber formation'. This is at the heart of my difficulty with this manuscript. There appears a confusion over interpretation of results - this is a major issue and needs to be addressed. In the introduction, around Line 83 you mention ZO-1. It would be nice to include some information about the significance of this relationship with ROK and MLCK. I also think the introduction could benefit from a figure explaining where ROK and MLCK fit into the whole process of tight junctions and permeability. Either that or look to increase the detail in the introduction a bit because I don't think the roles of ROK and MLCK and how they overlap or not, is completely clear in the way it is currently written. The first part of the results section reads like a mixture of Materials and Methods, results and discussion. One might want to consider editing this a bit and strip it down to just reporting the results. I find figure 1 slightly unconvincing because some cells somewhat follow the pattern described and others don't - it appears random. Is there anyway to quantify this effect so you can illustrate the shift more easily? How effective are the drugs you are using - can you measure activity in any other way to confirm the drugs are doing what they are supposed to do? Line 233: Why no comment about the 96-hour timepoint? If it isn't relevant remove from the figure but if it is relevant, it's worth commenting on because it seems to be the only timepoints where differences occur. Figure 2B: On line 245 it states that ML7 didn't affect DV-induces permeability and yet in figure the values for ML7 are equivalent to mock and seem to be quite different to DV alone. Please explain. Figure 2A+B: Do you have any statistics to check for significance? Line 265-269: True for ML7, not for Y27632 at 24h. Not sure about any time point really because there are no statistics, which means you can't really make the statement on line 268 with any confidence. Line 270 - 294: The first thing that should be done with any drug is to test its cytotoxicity and identify an appropriate concentration to use. Was this done? Consider moving this section to the beginning of the results if so. Line 285: Please show this data, it is important. Do the drugs you are using have any off-target effects? Line 290-295: This is discussion not results. Also, considering there are no statistics associated with figure 3B and that at 72hpi virus produced is not different from ML7 treated cells, which doesn't cause an increase in cell death, this link is difficult to argue for. Line 306: Why such a low MOI if this is identified as an issue? Line 310 and figure 5: In the line you give the 50% value based on a method described in the figure legend - please put this method into the materials and methods section not the figure legend. Also, this should be labelled Fig 4. Line 315: Why do you think it is a gene expression issue? Could it not be a translation issue or increased protein degradation just as easily? Figure 5C: What was the standard used to calculate copy number? The figure legend needs updating to include (C). Y27632 appears to increase copy number at 72h compared to DV alone. Line 326-331: Save this for the discussion. What you state here doesn't match with the results - yes, you see reduced genome copy number and reduced protein but in figure 3 there isn't a statistically significant difference between DV alone and DV+ML7. Similar for the other drug. Line 353: This conclusion is not supported by the evidence presented, please consider revision of this or add further evidence to confirm it. Line 359: Where is this 3-fold reduction? I don't see that in figure 3. Line 363: Figure 5 doesn't show an excessive build up of protein and RNA Line 369: There is no strong evidence presented to support that virus release is affected. Line 375: I'm not convinced the evidence presented here properly supports this statement. Line 389: Why prior? Could it be directly affecting genome replication? Line 393: Would it be worth discussing that fact that Y27632 increases permeability and cell death? Also is there a significance to the fact that both apoptosis and necrosis appear to increase (Figure 3 B)? Line 402: neither drug prevents virus replication. Replication may be reduced, but it is not prevented. Separate comment: Throughout the manuscript it is maintained that the drugs have no effect on infectivity, but the drug is never added prior to initial infection, so how do you really know?

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    No: Not applicable - no animals or humans involved

  7. Version published to 10.1099/acmi.0.000845.v1 on Access Microbiology