m6A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB

Chia-Chieh Chu; Bei Liu; Raphael Plangger; Christoph Kreutz; Hashim M. Al-Hashimi

doi:10.1371/journal.pone.0224850

Peer Review History

Original SubmissionOctober 19, 2019
5 Nov 2019 Decision Letter - Joanna Sztuba-Solinska, Editor PONE-D-19-28951 m6A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB PLOS ONE Dear Dr. Hashim M. Al-Hashimi, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has great 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. Your manuscript has been revised by four reviewers with two of them indicating minor changes indicated below. We would appreciate receiving your revised manuscript by December 06, 2019. 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. 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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 Reviewer #4: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: N/A Reviewer #3: I Don't Know Reviewer #4: 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 Reviewer #4: 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 Reviewer #4: 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: The effect on structure of methylation of A68 in the stemloop IIB of the HIV RRE has been studied in this report using a variety of physical-chemical methods. All the experiments were conducted in vitro with purified RNA substrates. The study clearly shows that the methylation causes no significant change in any of the parameters measured in any of the studies. It also shows that greater effects of methylation can be seen when measurements are conducted in the absence of magnesium, underscoring the importance of carrying out measurements under more physiological conditions. Intriguingly, the authors did show a slight (2 fold) change in the binding of the Rev-ARM to the structure containing the methylation. Contrary to other reports the effect was actually a weakened binding. The authors point out that this is more consistent with what would be expected given the physical effects expected from the methylation. As the authors carefully point out, these cannot studies do not rule out that methylation may effect the full-length RRE struture, full-length Rev protein binding or the binding of other cellular factors differently. This is a weakness of this study but in so far as the authors address this issue clearly and the data presented here is sound, this study seems to fit the criteria needed to be acceptable for PLOS One. Reviewer #2: The authors present evidence that methylation has minimal effects on the properties of stem IIB of HIV-1 RRE. They use several biophysical techniques in this analysis. This conclusion is interesting and potentially significant. Reviewer #3: Chia-Chieh Chu and collaborators present a controversial article where they claim that m6A has little impact on the HIV-1 RRE stem IIB structure and in a viral protein binding proprieties. The authors conclude by three biophysical experiments: optical melting experiments, NMR and binding assays that the m6A modification in the position 68 of the the Rev response element is not sufficient to produce significant changes in its conformation. Chu et al, also analyze a functional motif in Rev, one of its best-known proteins that bind RRE and they conclude that m6A does not affect its binding properties. This is a very interesting research article that prevent from dogmas since contradicts some of the literature cited. However, authors need to consider that discarding or dismissing another study requires more evidence. They need to soften some or their conclusions due to the nature of their in vitro studies. They lack description of some important controls for example: in all experiments, how do they make sure the correct “natural” folding of the RRE RNA? How do they know that all the molecules are folded in the same way in vivo? Would it be necessary to consider in vivo crosslinked samples in controls to validate their hypothesis? How do they measure the Rev-RRE association? Finally, they emphasize that Mg+ and the sequence context impact on m6A-RRE RNA structure, but they did not perform any experiments related to the sequence context. More experiments in this matter are necessary to support this conclusion of a less strong sentence needs to be changed to soften this conclusion. In line 256 they establish that “…m6A slightly weakens binding to the Rev-ARM peptide.” Could it be that “slightly” difference in the cell is catalytically important? In my opinion, this paper contains well documented and reliable experiments, it is very well constructed, detailed and has a didactic approach. However strong affirmations i.e. line 286 “This underscores the importance of studying the impact 287 of m6A modifications under conditions that mimic the physiological cellular environment.” Are not congruent with the data presented and need to be state in an ease way. Reviewer #4: There is some controversy in the field regarding the role of m6A methylation in HIV replication. In particular, the role of m6A methylation sites within the HIV Rev response element (RRE) stem IIB, which have previously been suggested to enhance binding to the HIV REV protein, which in turn enhances REV-mediated viral mRNA nuclear export. Several studies have failed to identify these proposed m6A sites using m6A-seq approaches. Chu et al., provide convincing evidence using a range of biophyisical approaches, such as UV melting and NMR-based approaches, to show that m6A methylation does not affect the structure of RREIIB. Moreover, flourescence aniosotrophy was used to suggest that m6A in this region actually weakens REV binding. Specific comments. 1. The introduction could be extended to include other examples of how it can stabilise RNA structures and is implicated in various other RNA processing events. 2. There is no mention of m6A readers. 3. A better explanation of why Mg2+ is brought into the experiments and its significance is required throughout the paper. 4. Line 207 - needs amending 5. The flourescence aniosotrophy experiment is interesting, but very brief, this is a complete opposite to the original finding. Therefore, it needs a confirmatory experiment, as this is just based on a REV-ARM peptide to the RREIIB. ****** 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: Yes: Gabriela Toomer Reviewer #4: 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. 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Revision 1
24 Nov 2019 Author Response Reviewer #1: “The effect on structure of methylation of A68 in the stemloop IIB of the HIV RRE has been studied in this report using a variety of physical-chemical methods. All the experiments were conducted in vitro with purified RNA substrates. The study clearly shows that the methylation causes no significant change in any of the parameters measured in any of the studies. It also shows that greater effects of methylation can be seen when measurements are conducted in the absence of magnesium, underscoring the importance of carrying out measurements under more physiological conditions. Intriguingly, the authors did show a slight (2 fold) change in the binding of the Rev-ARM to the structure containing the methylation. Contrary to other reports the effect was actually a weakened binding. The authors point out that this is more consistent with what would be expected given the physical effects expected from the methylation. As the authors carefully point out, these cannot studies do not rule out that methylation may effect the full-length RRE struture, full-length Rev protein binding or the binding of other cellular factors differently. This is a weakness of this study but in so far as the authors address this issue clearly and the data presented here is sound, this study seems to fit the criteria needed to be acceptable for PLOS One.” We thank the reviewer for his/her comments. Reviewer #2: The authors present evidence that methylation has minimal effects on the properties of stem IIB of HIV-1 RRE. They use several biophysical techniques in this analysis. This conclusion is interesting and potentially significant. We thank the reviewer for his/her comments. Reviewer #3: 1) “Chia-Chieh Chu and collaborators present a controversial article where they claim that m6A has little impact on the HIV-1 RRE stem IIB structure and in a viral protein binding proprieties. The authors conclude by three biophysical experiments: optical melting experiments, NMR and binding assays that the m6A modification in the position 68 of the the Rev response element is not sufficient to produce significant changes in its conformation. Chu et al, also analyze a functional motif in Rev, one of its best-known proteins that bind RRE and they conclude that m6A does not affect its binding properties. This is a very interesting research article that prevent from dogmas since contradicts some of the literature cited. However, authors need to consider that discarding or dismissing another study requires more evidence. They need to soften some or their conclusions due to the nature of their in vitro studies. They lack description of some important controls for example: in all experiments, how do they make sure the correct “natural” folding of the RRE RNA? How do they know that all the molecules are folded in the same way in vivo? Would it be necessary to consider in vivo crosslinked samples in controls to validate their hypothesis? How do they measure the Rev-RRE association?” The structure of RRE stem IIB has been the subject of numerous investigations in vitro by X-ray, NMR, SAXS and also more recently in the context of much larger RRE fragments using chemical probing data. In all cases, the structure of RREIIB has been determined to be the same as that observed under our NMR conditions as elaborated in ref 28. We have clarified this point on page 9 on line148 by stating that “Prior X-ray [ref 42, 43], NMR [ref 44, 45], SAXS [ref 46] as well as chemical probing data on larger fragments of the HIV genome (~350 nt) [ref 47] indicate that RREII stem IIB adopts the predominant secondary structure shown in Fig 1A.” We agree with the reviewer that we cannot rule out that the binding properties may differ in vivo and this is why we noted in the original submission on line 292 that “Our data cannot rule out that the exposed and accessible m6A enhances binding in the context of full-length Rev and RRE or promotes RNA export through the recruitment of other host export factors.” We agree with the reviewer that the structural and dynamic properties of RREIIB may differ in vivo. In a recent study [ref 29], we showed that the dynamic properties of RREIIB determined in vitro are very similar to the dynamic properties measured in cells. This argues against a major difference between the two environments at least for unmodified RREIIB. Nevertheless, to soften the language and emphasize this possibility, we added the following sentence to our discussion on page 16 on line 296, “It is also possible that the structural and dynamic properties of m6A modified RREIIB differ in vivo relative to in vitro. A recent study [ref 29] showed that the key dynamic properties of unmodified RREIIB are similar in vitro and in cells. Further studies are needed to examine the behavior of m6A modified RREIIB in cells. “ 2) Finally, they emphasize that Mg+ and the sequence context impact on m6A-RRE RNA structure, but they did not perform any experiments related to the sequence context. More experiments in this matter are necessary to support this conclusion of a less strong sentence needs to be changed to soften this conclusion. With regards to different sequence contexts, we were referring to other published studies cited in our manuscript [refs 31, 32, 36, 37] which showed that m6A has different effects on RNA stability depending on sequence context and in some cases on the presence/absence of Mg2+. We have clarified this by adding the following sentence at the end of the introduction: “For example, while m6A has been shown to destabilize canonical duplexes [ref 31, 32], it can stabilize junctional A-U base pairs in a Mg2+ and secondary structure dependent manner [ref 37] as well as in contexts in which the m6A is in a dangling end [ref 32].” 3) In line 256 they establish that “…m6A slightly weakens binding to the Rev-ARM peptide.” Could it be that “slightly” difference in the cell is catalytically important? In my opinion, this paper contains well documented and reliable experiments, it is very well constructed, detailed and has a didactic approach. However strong affirmations i.e. line 286 “This underscores the importance of studying the impact 287 of m6A modifications under conditions that mimic the physiological cellular environment.” Are not congruent with the data presented and need to be state in an ease way. We appreciate the comment made by the reviewer. We agree that we need to be cautious not to over interpret the data measured in vitro as the behavior could change in vivo. In the original submission, we did acknowledge that the in vivo environment could alter the behavior. In the revised manuscript, we made additional changes to soften the language. In the abstract on line 22, we replaced “unlikely to substantially alter the RRE-Rev interaction” with “m6A is unlikely to substantially alter the conformational properties of the RRE.” so that the focus is on the impact of m6A on the conformational properties. On line 65, we now note, “The results indicate that if RREIIB is m6A modified, it is unlikely to substantially change the conformational properties of RRE although we cannot rule out that small changes in the conformational properties could modulate Rev-RRE binding in vivo.” In the revised manuscript, we also edited the following sentence in the discussion section on line 304 “This underscores the importance of studying the impact of m6A modifications in the presence of Mg2+ as in a more physiological conditions.” Reviewer #4: There is some controversy in the field regarding the role of m6A methylation in HIV replication. In particular, the role of m6A methylation sites within the HIV Rev response element (RRE) stem IIB, which have previously been suggested to enhance binding to the HIV REV protein, which in turn enhances REV-mediated viral mRNA nuclear export. Several studies have failed to identify these proposed m6A sites using m6A-seq approaches. Chu et al., provide convincing evidence using a range of biophyisical approaches, such as UV melting and NMR-based approaches, to show that m6A methylation does not affect the structure of RREIIB. Moreover, flourescence aniosotrophy was used to suggest that m6A in this region actually weakens REV binding. Specific comments. 1. The introduction could be extended to include other examples of how it can stabilise RNA structures and is implicated in various other RNA processing events. We thank the reviewer for this suggestion. We added text to refer to specific examples where m6A can stabilize RNA (see response to comment 2 by Reviewer 3). 2. There is no mention of m6A readers. In the revised manuscript, we added the following sentence in the introduction on line 44 “Another study employing distinct cell lines and mapping methods did not observe these m6A sites on RREIIB suggesting that m6A can enhance HIV-1 replication and mRNA expression through recruitment of the m6A reader protein YTH-domain containing family (YTHDF) [ref 21]. A third study found m6A in RRE but did not identify the specific site [ref 18]. The study proposed that the YTHDF proteins inhibit HIV-1 replication and infection by blocking viral reverse transcription.” 3. A better explanation of why Mg2+ is brought into the experiments and its significance is required throughout the paper. We thank the reviewer for the suggestions. In the revised manuscript, we added the following sentences on line 142 “All experiments were performed in the presence of 3 mM Mg2+ unless stated otherwise. This is important given the impact of Mg2+ on RNA folding and dynamics [ref 41] and also given recent studies showing that the impact of m6A on RNA structural dynamics and stability can depend on Mg2+ [ref 37]. 4. Line 207 - needs amending In the revised manuscript, we edited the following sentence in the result section in line 219 (previous line 207) “We recently showed that RREII transiently adopts two low-abundance alternative secondary structures (‘excited states’, ES) referred to as ES1 and ES2 (Fig 3A).” 5. The flourescence aniosotrophy experiment is interesting, but very brief, this is a complete opposite to the original finding. Therefore, it needs a confirmatory experiment, as this is just based on a REV-ARM peptide to the RREIIB. The binding affinity for m6A modified was only slightly weakened relative to unmodified RREIIB especially when considering the uncertainty. So our results are best described not as opposite to what was reported previously in ref 20; rather we did not see any evidence for tighter binding with m6A modified RREIIB. We revised the manuscript to soften the language when making this point on page 14 on line 268, “the binding affinity for m6A modified was only slightly weakened relative to unmodified RREIIB especially when considering the uncertainty”. In addition, we note that no Kd was measured in the prior study [ref 20]. Rather the tighter affinity was deduced based on ~2-3 fold decrease in pull down of RRE RNA when knocking down two subunits (METTL3/METTL14) from the methyltransferase complex responsible for producing m6A. To address the reviewer comment, we added the following sentence in the discussion on line 292, “Our data cannot rule out that the exposed and accessible m6A enhances binding in the context of full-length Rev and RRE or promotes RNA export through the recruitment of other host export factors, and that this in turn gives rise to the reported ~2-3 fold decrease in HIV viral RNA pull down using Rev when knocking down METTL3/METTL14 responsible for producing m6A [ref 20] ”. Editorial 1. We noticed you have some minor occurrence of overlapping text with your previous publication, which needs to be addressed: - https://academic.oup.com/nar/article/47/13/7105/5519168 In your revision ensure you rephrase any duplicated text outside the methods section. In the revised manuscript, we edited the text of fluorescence polarization assays in the methods section to avoid overlapping text. 2. Thank you for including your competing interests statement; "I have read the journal's policy and the authors of this manuscript have the following competing interests: H.M.A. is an advisor and holds an ownership interest in Nymirum Inc., an RNA- based drug discovery company. " Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. In the revised manuscript, we included “This does not alter our adherence to PLOS ONE policies on sharing data and materials.” in Conflict of interest statement section. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0224850.r002
27 Nov 2019 Decision Letter - Joanna Sztuba-Solinska, Editor m6A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB PONE-D-19-28951R1 Dear Dr. Al-Hashimi, 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, Joanna Sztuba-Solinska, Ph.D. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0224850.r003
Formally Accepted
4 Dec 2019 Acceptance Letter - Joanna Sztuba-Solinska, Editor PONE-D-19-28951R1 m⁶A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB Dear Dr. Al-Hashimi: 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 Dr. Joanna Sztuba-Solinska Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0224850.r004

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