Glutathione contributes to efficient post-Golgi trafficking of incoming HPV16 genome

Shuaizhi Li; Matthew P. Bronnimann; Spencer J. Williams; Samuel K. Campos

doi:10.1371/journal.pone.0225496

Peer Review History

Original SubmissionSeptember 25, 2019
22 Oct 2019 Decision Letter - Craig Meyers, Editor PONE-D-19-26859 Glutathione Contributes to Efficient Post-Golgi Trafficking of Incoming HPV16 Genome PLOS ONE Dear Dr. Campos, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We would appreciate receiving your revised manuscript by Dec 06 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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If you would prefer to remove your manuscript from collection consideration, please specify this in the cover letter. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: 488 This work was supported by grant 1R01AI108751-01 from the National Institute for 489 Allergy and Infectious Diseases. We are grateful to Dr. Martin Müller for the K4-L220-38 490 monoclonal antibody, Dr. Martin Sapp for the L1-7 monoclonal antibody, Dr. Michelle 491 Ozbun for the anti-HPV16 polyclonal antibody, Dr. Michael Barry for the HAdV5 vector, 492 Dr. Chris Buck for the 293TT and Dr. Anne Cress for the HaCaT cells. We thank Patty 493 Jansma of the UA ORD Imaging Core-Marley, and Paula Campbell and John Fitch of 494 the UACC/ARL Cytometry Core Facility, which is funded by a UA Cancer Center 495 Support Grant (CCSG - CA 023074). We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "SKC is supported by grant 1R01AI108751-01 from the National Institute for Allergy and Infectious Diseases, https://www.niaid.nih.gov/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 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 Reviewer #3: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Cellular entry of papillomaviruses is a complex process that is only partly understood. A critical stage of entry involves the penetration of a portion of L2 across the endosomal membrane to facilitate trafficking of vesicles containing the viral genome to the nucleus. In this paper, Li et al show that inhibition of the cellular glutathione system through drug treatment of knockdown of GSH-generating enzymes results in less efficient penetration of L2 into the cytoplasm, trafficking of the virus to the nucleus, and consequently virus infection. This is a nice paper, well executed and well presented, and the data are convincing. A few points/questions should be addressed: 1. The authors make the point (lines 326-330) that BSO treatment does not affect the cell cycle. They say that there was “a subtle but statistically insignificant expansion of S phase upon BSO treatment.” If the expansion is not statistically significant, then they cannot say that there is an expansion. It also does not help their case to claim that there is one. Better to say something like “no statistically significant changes in cell cycle profiles were observed”, and leave it at that. 2. In figure 3C, there seems to be an increase in the levels of the 25kD degraded L1 band in BSO vs control. Was this consistently observed? The authors should quantify their western results across several blots. 3. The graph in Fig 4D is kind of hard to read with the little symbols. It would be better to indicate the things being quantified with labels under the X axis. 4. In the discussion, the authors discuss ways that GSH might affect vesicular trafficking. This may important, but their own data in Fig 5 show that penetration of L2 C terminus into the cytoplasm is the key step. Failure to translocate L2 would be sufficient to explain the failure in trafficking of HPV-containing vesicles. In that light, the authors should discuss more about what is known about that translocation step. How might cellular redox affect it? What is the relationship between redox in the cytoplasm and redox in the endosomal lumen, where L2 would be present prior to translocation? Does L2 have redox-sensitive cysteines? 5. Would knockdown of the GSH production machinery affect the levels of NADPH? Is it known what effect high NADPH might have on the cell? Reviewer #2: Overall, this manuscript provides relevant and interesting insight into what has been an experimentally challenging question – that of understanding, at a molecular level, how HPV virions enter the cell and begin their life cycle. While the insight now provided regarding how the virions exit the trans-Golgi is somewhat general, it does provide a useful framework and sets the stage for a more detailed molecular explanation in the future. Specific comments are noted below. • A figure showing virion entry and trafficking, highlighting the areas where glutathione/redox is likely to be important, would help the reader to navigate the manuscript. • Have the authors definitely established that it is the specific concentrations of the actual GSH molecule that are required, or could the requirement be something more general, such as redox potential? It may be possible to provide experimental evidence one way or the other; alternatively, perhaps the language could be broadened somewhat. (For example, see page 20, lines 443-445). Reviewer #3: This manuscript from the Campos group highlights a key role for aspects of the cellular redox system in HPV16 infection. Using compounds and siRNA depletion of key cellular factors coupled to established (and well controlled) assays they demonstrate that the role of these factors appears to occur in post-Golgi trafficking. The study is of interest to those working out the convoluted infection process of HPV. The data are tight and the manuscript well written. I do not see a need to ask the authors to perform more experiments when the present study is sufficient. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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 Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0225496.r001
Revision 1
31 Oct 2019 Author Response Response to Reviewers (responses in red) Reviewer #1: Cellular entry of papillomaviruses is a complex process that is only partly understood. A critical stage of entry involves the penetration of a portion of L2 across the endosomal membrane to facilitate trafficking of vesicles containing the viral genome to the nucleus. In this paper, Li et al show that inhibition of the cellular glutathione system through drug treatment of knockdown of GSH-generating enzymes results in less efficient penetration of L2 into the cytoplasm, trafficking of the virus to the nucleus, and consequently virus infection. This is a nice paper, well executed and well presented, and the data are convincing. A few points/questions should be addressed: 1. The authors make the point (lines 326-330) that BSO treatment does not affect the cell cycle. They say that there was “a subtle but statistically insignificant expansion of S phase upon BSO treatment.” If the expansion is not statistically significant, then they cannot say that there is an expansion. It also does not help their case to claim that there is one. Better to say something like “no statistically significant changes in cell cycle profiles were observed”, and leave it at that. response: Thank you for raising this concern but we feel that these subtle effects of BSO may indeed be biologically relevant, even if they are not statistically “significant” based on the commonly used p-value threshold of 0.05. We feel it’s better to be upfront in reporting these differences and say they were not significant rather than to ignore or dismiss them, especially because these subtle trends were seen across multiple independent experiments. The text has been slightly modified to better reflect this perspective. 2. In figure 3C, there seems to be an increase in the levels of the 25kD degraded L1 band in BSO vs control. Was this consistently observed? The authors should quantify their western results across several blots. response: Thank you for pointing this out. Further analysis of multiple replicates do indeed reveal a significant difference in degraded L1 at this 2h time point. We have performed densitometry of these reps and include that analysis as new panels in figure 3. Text has been modified accordingly. 3. The graph in Fig 4D is kind of hard to read with the little symbols. It would be better to indicate the things being quantified with labels under the X axis. response: We have modified figure 4, and directly labeled the X-axis as suggested. 4. In the discussion, the authors discuss ways that GSH might affect vesicular trafficking. This may important, but their own data in Fig 5 show that penetration of L2 C terminus into the cytoplasm is the key step. Failure to translocate L2 would be sufficient to explain the failure in trafficking of HPV-containing vesicles. In that light, the authors should discuss more about what is known about that translocation step. How might cellular redox affect it? What is the relationship between redox in the cytoplasm and redox in the endosomal lumen, where L2 would be present prior to translocation? Does L2 have redox-sensitive cysteines? response: Our data indicated failure of L2/vDNA to traffic post-Golgi. This could be due to failure of L2/vDNA to bind mitotic chromosomes or failure of L2/vDNA to egress/traffic from the vesiculating Golgi. We added a bit to what is known about the post-Golgi trafficking and translocation step in the discussion, lines 464-475. 5. Would knockdown of the GSH production machinery affect the levels of NADPH? Is it known what effect high NADPH might have on the cell? response: We did not directly look at NAD+/NADH (or NADP/NADPH) levels, but GSH depletion certainly affects these redox couples which may indeed affect many aspects of cellular physiology including DNA damage (via NAD+ dependent PARP activity), survival, and ROS responses. All these probable effects add to the challenges of understanding the molecular basis for the observed phenotype. We chose not to comment or speculate too much in this regard. Reviewer #2: Overall, this manuscript provides relevant and interesting insight into what has been an experimentally challenging question – that of understanding, at a molecular level, how HPV virions enter the cell and begin their life cycle. While the insight now provided regarding how the virions exit the trans-Golgi is somewhat general, it does provide a useful framework and sets the stage for a more detailed molecular explanation in the future. Specific comments are noted below. • A figure showing virion entry and trafficking, highlighting the areas where glutathione/redox is likely to be important, would help the reader to navigate the manuscript. response: We have added a final summary figure 6 based on the recommendation. • Have the authors definitely established that it is the specific concentrations of the actual GSH molecule that are required, or could the requirement be something more general, such as redox potential? It may be possible to provide experimental evidence one way or the other; alternatively, perhaps the language could be broadened somewhat. (For example, see page 20, lines 443-445). response: We currently cannot say whether it’s the GSH molecule itself, the GSH/GSSG redox potential, or downstream effects on other redox couples. We have broadened the language in the discussion to reflect this, lines 471-477. Reviewer #3: This manuscript from the Campos group highlights a key role for aspects of the cellular redox system in HPV16 infection. Using compounds and siRNA depletion of key cellular factors coupled to established (and well controlled) assays they demonstrate that the role of these factors appears to occur in post-Golgi trafficking. The study is of interest to those working out the convoluted infection process of HPV. The data are tight and the manuscript well written. I do not see a need to ask the authors to perform more experiments when the present study is sufficient. Attachments Attachment Submitted filename: response letter.docx https://doi.org/10.1371/journal.pone.0225496.r002
7 Nov 2019 Decision Letter - Craig Meyers, Editor Glutathione Contributes to Efficient Post-Golgi Trafficking of Incoming HPV16 Genome PONE-D-19-26859R1 Dear Dr. Campos, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Craig Meyers, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0225496.r003
Formally Accepted
12 Nov 2019 Acceptance Letter - Craig Meyers, Editor PONE-D-19-26859R1 Glutathione Contributes to Efficient Post-Golgi Trafficking of Incoming HPV16 Genome Dear Dr. Campos: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Craig Meyers Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0225496.r004

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