PONE-D-21-35236Evidence for a long-r ange RNA-RNA interaction between ORF8 and the downstream region of the Spike polybasic insertion of SARS-CoV-2PLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: I Don't Know

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: # Review for PONE-D-21-35236

Evidence for a long-r ange RNA-RNA interaction between ORF8 and the downstream region of the Spike polybasic insertion of SARS-CoV-2

Filipe Pereira and Amirhossein Manzourolajdad

# Summary

The article summarizes the investigation of potential long-range RNA-RNA interactions (RRIs) within the SARS-CoV-2 genome and related sequences in SARS-CoV-1. In details, the study focuses on the genomic subregion 23600-24200 that is part of the Spike gene. The region is defined by the intra-molecular helix formed by a SARS-CoV-2-specific 12nt insert located at the beginning of the region that forms stable base pairing with the region's end.

The authors performed IntaRNA-based RRI prediction to screen for RRIs that interlink the genomic subregion with other parts of the genome respectively for both viruses. Two high-scoring interactions were identified, i.e. a stable and conserved RRI of the region's end with ORF1ab and a non-conserved RRI that interlinks in SARS-CoV-2 the beginning of the region with ORF8.

Following the headlines of the Results section, the main findings by the authors are (1) locally stable structure within the region (2) the two stable interactions mentioned above and (3) support for the formation of the Spike-ORF8 RRI via covariation analyses.

# General remarks

I have couple of central issues with this manuscript in its current form.

## (1) 12 nt insert irrelevant and obscures the focus and informative value

Beside the definition of the genomic subregion of interest, the 12 nt insert is eventually unimportant for any statement supported by the study (beside observing that S1 stem loop is stable with and without the insert).

The authors write themselves "The contribution of the 12-nt insert [...] is still unclear".

Thus, I have to ask: why then using the 12-nt fragment as such a rigid anchor for the study? The authors use about 2/3 of the Spike gene somewhat at random (based on the 12nt and its structure formation) instead of the whole gene! The rational for taking the whole could be the same, i.e. "12-nt insertion is inside", but now the SARS-CoV-1 subregion is clearly defined (as the related full gene sequence) and not based on the mapping of a substructure region...

Using just a subregion of the gene

(a) causes problems with local structure prediction AT LEAST at the ends of the regions if not overall,

(b) has thus implications on RRI prediction, since IntaRNA incorporates the accessibility of interacting subregions, which is based on local structure prediction, and

(c) restricts the insights of the study to a hypthetical local fragment rather than a valid mRNA-like molecule.

## (2) mfe structures based on global folding are no local structure signal

Using minimum free energy (mfe) estimates based on GLOBAL structure prediction (i.e. allowing base pairing over the range of the RNA) and respective structure plots to discuss local structure formation is inappropriate. The mfe is only ONE possible stable structure and strongly depends on the underlying thermodynamic model. Mfe prediction gets decreasingly reliable (in its details) the longer the RNA. Thus, instead of individual base pairing (of the presented mfe structure), LOCAL base pair probabilities (eg visualized via dot plots) are a much sounder tool to investigate local structure formation of RNAs or genomic regions. And if the details of base pairing of of no importance, even local unpaired probabilities (related to the accessibility values used by IntaRNA), are simple tool to identify locally (un)structured regions etc. without detailing base pairing.

When presenting mfe predictions of different tools (Fig-1) that are based more or less on the same thermodynamic energy model, no further insight is gained...

Furthermore, structure representations (Fig-1, Fig-4) are

-) so small that they are not readable in print

-) due to the latter, coloring is completely lost in print

-) eventually unimportant and not discussed in details for the RRI prediction

## (3) There is no covariation as far as I can see...

Please check Fig-1a of

https://academic.oup.com/bioinformatics/article/36/10/3072/5729989

the only thing I observe in the provided data (Fig-3) is VARIATION, i.e. only one side of the base pair shows variants...

The whole covariation part has several flaws..

(a) the authors use the concatenated interacting subsequences as an input to the CO-FOLDING BASED R-scape software... This is terribly wrong, since no LINKER sequence spacing the two FRAGMENTS is used. Without it, the helix close to the of the interaction that is close to the point of concatenation cannot be formed due to STERICAL reasons respected by the structure prediction software... Thus, the co-variation study misses eventually 5 base pairs and even more important, the Spike region is ONE TOO SHORT! missing the final interacting U...

?!?

If done that way, ie. using the co-folding based approach, one has to insert a linker region of at least 3-5 nt, eg. poly-A or if possible poly-N, to allow for enough flexibility that the concatenated ends of the sequences can form a structure. Or why not extending the subregion range by 5nt in both directions? would be simple and avoid this central error..

(b) who does the counts in Fig-3b relate to the substitution count in Table-1? The authors refer to the manual of R-scape, which is in no way a proper scientific explanation, even more so since these counts are central for selecting pairs and position within the argumentation of the authors..

(c) the substitution counts incorporate AMBIGOUS nucleotides (Fig-3b)... ?!? What about sequencing errors etc. as a source of such ambiguity? For other parts of the study such subsequences were excluded, why not here?

(d) the authors MISS an important point: the IntaRNA predictions are annotated with based pairs that are likely to be formed first, the seed base pairs represented by "+" within the plot. These base pairs seem to correlate with LOW subsitution scores, which I would find a supportive and good sign! That is low sequence variation in the important sub-RRI!

Thus, an investigation and relation of VARIATION with RRI formation would be more appropriate than a co-variation study that shows there is no co-variation..

## (4) Why showing bi-/tri-molecule co-folding results?

I find no rational for nor any additional insights from the bi-/tri-molecule folding part of the manuscript. The description of the assembly of the tri-molecule prediction from bi-molecule graphs is very vague.

Maybe I missed it, but what's the point? Fig-4a shows the same RRI patterns as Fig-2a. The lack of structure relation of Fig-4b and Fig-2b can/will have multiple possible reasons that are not discussed at all..

If you are interested in co-formation of multiple RRIs, why not using a constraint IntaRNA prediction? Therein you can mark regions that are involved in base pairing (eg. an RRI) and you will get corrected predictions for the rest of the molecule EXCLUDING the ballast of local intra-molecular mfe structure (see (2)).

But eventually, that wasnt even the point, or was it? I am confused by this part..

## (5) No suboptimal RRIs investigated/shown/discussed ...

For RRIs, the same holds as discussed for (2). While base pairing probability prediction is much harder for RRI prediction compared to intra-molecular structure prediction, one can relate to suboptimal interactions as a substitute to assess the structural variety. This is needed at multiple parts within this manuscript:

(a) the extremely long UTF interaction in Fig-2b is most likely an artifact of the summation-based prediction scheme. Simply spoken: the more base pairs the better until accessibility penalties overweight the gain.

BUT: did you check suboptimal interactions of the region? I would expect much shorter sub-RRIs with similar stability (i.e. energy).

(b) Furthermore did you check if a similar ORF8 interaction is among the suboptimals of the SARS-CoV-1 predictions? Since the ORF8 SARS-CoV-2 RRI shows a lower energy that the UTF-RRI, the chance is high that it is "hidden" there...

# Conclusion

Given the length of my remarks and the bulk of flaws, I cannot recommend the manuscript in its current form for publication.

I hope the authors take the time to really reshape and rework this study before resubmitting as I took the time to point out the weaknesses.

Reviewer #2: Prior studies have discovered many long-range RNA-RNA interactions within the genomes of positive-strand RNA viruses, which have functional roles in fundamental viral processes. In this submission, Pereira and Manzourolajdad analyze SARS-CoV-2 genomic sequences from the GISAID database. They report a long-range interaction of a region containing a 12-nt insertion in the Spike protein region that is not present in SARS-CoV. They use standard methods to analyze RNA sequence data, and find locally stable structural features downstream of the insertion region, evidence for potentially functional interactions with ORF8 region, and some evidence for sequence covariation in the interacting regions. These findings can be useful to understand the biology of SARS-CoV-2 and develop therapies. Eventually, experimental data will be needed to ascertain the presence of these interactions in vivo and its functional significance. However, that may be beyond the scope of the present study. I have focused my comments on the computational analysis, which I hope the authors will consider addressing.

1. The 12 nt insertion site is 23,603-23,614 of the genome. The authors searched for long range interactions of any SARS-CoV-2 region with the region 23,600-24,107 and 23,917-24,118, i.e., only downstream region of the insertion site. Shouldn't equally long stretches upstream of the insertion site be considered as well? It is not clear to me why the present study has focused on the downstream region only.

2. Similar to my previous question, it is not clear why the overlapping regions 23,600-24,107 and 23,917-24,118 were analyzed separately. It will be helpful if the authors can comment on this.

3. The developers of IntaRNA have shown that integrating experimentally obtained chemical probing data with IntaRNA can significantly improve RNA-RNA interaction prediction. Since such data is available for SARS-CoV-2, have the authors considered using it in their analysis?

4. In the second subsection of the Results, the authors report long-range interaction predictions from IntaRNA. Are these all the predictions from IntaRNA or have the authors reported the subset of the most significant predictions? It might be worthwhile to add a Supplementary table listing all the interactions that meet a reasonable significance cutoff.

5. In the paragraph above Table 1, it seems there is a typo in the second sentence. For the region 23,675-28,045, the table shows n=92 while the text says n=2.

6. There are many grammatical mistakes scattered throughout the manuscript, which should be corrected, if necessary using free software such as Grammarly.

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PONE-D-21-35236R1Evidence for a long-range RNA-RNA interaction between ORF8 and Spike of SARS-CoV-2PLOS ONE

Dear Dr. Manzourolajdad,

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.

The revised manuscript should address all the critical points raised by the important reviewer.

Please submit your revised manuscript by Jun 05 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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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Comments to the Author

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Reviewer #1: (No Response)

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Reviewer #1: Partly

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Reviewer #1: I Don't Know

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Reviewer #1: No

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Reviewer #1: # Review for PONE-D-21-35236-R1

Evidence for a long-range RNA-RNA interaction between ORF8 and Spike of SARS-CoV-2

Okiemute B. Omoru, Filipe Pereira, Sarath Chandra Janga, Amirhossein Manzourolajdad

# Summary

In short, the authors want to identify putative long-range RRIs within the SARS-CoV-2 genome that have high chance to be virus specific. To this end, they do a comparative investigation with SARS-CoV-1. Since there is a SARS-CoV-2-specific high-GC insert in Spike, the authors focus on this gene. Using a sliding window approach, putative interaction sites of Spike with other genomic regions are identified for both viruses. Since only SARS-CoV-2 shows a stable RRI with ORF8, the authors focus on the support of one of the predictions using structure prediction, base pair probabilities and (co-)variation analyses.

# General remarks

The revised work has positively sharpened the focus of the manuscript and provides more motivation for the whole endeavor. While improved, it still shows flaws and is still, in my opinion, of limited interest.

My major points are:

(*) There is no evidence anywhere that the identified RRIs are true "long-range" interactions!

The best one can speak of are "putative long-range" interactions, since (a) the whole identification and prediction is *fragment-based*, *in-silico* and without evidence that the full genome forms the interaction. It is also quite likely that the interaction is formed but only by (m)RNA fragments such that the whole hypothesis of the manuscript would be lost...

This is nowhere discussed within the manuscript!

Furthermore, both title and main contributions of the article need to be rephrased that way..

(*) The presented Pseudoknot structure prediction is neither local nor in line with the other used tools.

While it is hard to impossible to study or compare the dot-bracket-reported PK structures (Table 3), I find their presentation of no use and eventually wrong in the used context. First, the ProbKnot tool does a GLOBAL mfe prediction, with the limitations I discussed in the last review. Thus to infer local structure information from single mfes is, in my opinion, optimistic and wrong. Furthermore, all other used models within the study (IntaRNA and bp-prob computation) are based on nested structure models.. Thus, the base pair probs discussed along with the PK are using a different energy AND structure model and are thus hard to compare and a wrong intuition is triggered by the current text layout (namely that bp probs and PK structure are related).

Why using it at all? In the end, only a single minor crossing helix (5bp) is found and none for SARS-CoV-1. I doubt the relevance of this observation. Even more so while the authors have no mechanistic or whatsoever explanation or discussion of the observation (beside that it is observed).

To sanatize the course of the manuscript, I recommend to drop the PK part.

(*) Inconsistencies within the manuscript

- the Methods section still lists tons of tools that are not used in the current version

- the manuscript still referes to bifold predictions that are not present

- the 2nd and 4th paragraph of the introduction are mainly redundant

- the RRI visualizations (taken from IntaRNA) are using local fragment indices for Spike rather than the genomic positions, which makes it hard to follow and map the information. Just edit! (both in Fig-2 and supplement)

(*) Missing rationals

- the reason why the authors investigate the Spike-ORF8 RRI is only given within the discussion and one is lost wondering in the result section

- the use and interpretation of the AIC is no where to find and it stays unclear if the reported values are good or bad or how to interprete at all..

(*) Missing data

- the genome versions are not given (important since the reference genomes are undergoing some changes)

- the supplement lists the whole set of 2 million genome IDs but not the 200k used for the analyses

- how was the linear energy model derived? tool? library? handwritten?

(*) Overstating seed base pairs of RRIs

The "+" annotated bps in IntaRNA outputs are from stable subinteractions (of a used/default defined length, typically 7bp). Thus, these so called seed interactions are (in itself) stable enough to form (i.e. typically in unstructured regions) and thus likely to be starting points of the full RRI formation. Since an interaction can cover multiple such regions, all respective bps are annotated.

Currently, the manuscript describes these basepairs as "those that pair earlier than other base pairs", which is not true but again just a hypothesis.

Respective formulations should be amended respectively.

(*) Artifacts from subopt-limit

Since the authors limited the predicted suboptimals to 5 per fragment, the interaction atlas presented in Fig-1 is limited too. It could be that certain regions could interact with even more regions, which just dont pop up due to the hard "top-5" limit.

While this is no big drawback, it needs discussion. Even more so since the lack of predicted RRIs is a central point of discussion within both the result as well as discussion section!

(*) Suggestion: alpha via p-values

Just a suggestion that could improve the presentation. Given a large amount of genome-wide predictions (as done here) it is possible to estimate p-values for the energy scores (as presented on the IntaRNA webserver). The IntaRNA package even provides a respective script for computation.

The p-values could be used to set the alpha channel of the arcs within the circle plots to highlight highly probable RRIs and to distinguish them from weaker ones.

Currently, it is hard to say what interactions are stable. Even more so, since there is no general energy cut-off etc.

(*) Fig 4 not interpretable in printed version (and hard in pdf)

The dot plots are so small that dots are hard to spot or interprete/compare in print.

But even in pdf this is hard since only a pixel graphics is provided that cannot be zoomed without getting pixelated.

I suggest to move both figures in vector graphics format (PDF) to the supplement in full page width each to allow for detailed investigation.

The authors could present a respective cutout for the main manuscript if needed.

(*) Minor issues that caught the eye

- the text uses "Orf.." instead of "ORF.."

- Table 1 (and the supplement table) do not use "ORF8" but rather just "8" etc.

- Table 1 shows a red highlight not discussed in its caption

- "11th top hit within a total of 66" .. what 66? or is about the 69?

- "segments each corresponding to a particular viral strain" .. nope, each corresponds to a full genome sequencing (i.e. sample) but not necessarily strain!

- Table 2: lines Spike 23679-80 should be bold too

- it is not discussed that the CaCoFold predictions (Table 2 + Fig 3) miss the left-most RRI part from Fig-2, thus, rendering that RRI part less likely

- Fig-3 seems to be of low quality

- no vector graphics.. zoom in provides pixel art ..

- "contains five pseudoknots" .. NO, only ONE KNOT but "5 crossing base pairs". A BIG difference!

- Fig-4: it would be helpful to state in the caption that most likely base pairs are colored in red (for non-math readers)

- the text often states "SARS-CoV" instead of "SARS-CoV-1"

- Fig-4 caption "using the partition function" is a useless comment. better name the used tool.

- Fig-4 "shown in bold" .. better use "highlighted with a black bar". You can also annotate the same region on the y-axis (same coordinates) and draw horizontal/vertical lines at the respective bar ends to guide the eye to the important corridors within the plot

- "as well as well"

- it would be interesting to relate the S1 hairpin with the dot plot or annotate it(s position) within

- the supplementary figure needs a caption or the figure has to be extended to be self-explaining (what is relating to what and where)

(*) Carving out the core RRI based on the variation and stability investigation

Eventually, I think the authors miss a central outcome of the study or do not present it as such. While all the "long-range" part and the hopeful hypothesis of its impact on virulence, regulation etc. is quite speculative, the authors miss to highlight that the integration of RRI prediction and variation analyses strongly identifies the core of the putate Spike-ORF8 interaction. Namely the seed-region stretch 23679-23690 (Spike). The left part of Fig-2 is not predicted in Fig-3 and no variation is seen in this area.

Thus, one can conclude that the true RRI part (or at least the most likely part) is defined by that region and that it might be relevant (but maybe not exclusively for the RRI) since it is not mutated in both genes.

Why is it that most core conclusions of that manuscript are by reviewers?

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Reviewer #1: Yes: Martin Raden

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A Putative long-range RNA-RNA interaction between ORF8 and Spike of SARS-CoV-2

PONE-D-21-35236R2

Dear Dr. Manzourolajdad,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- 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.

Kind regards,

Danny Barash

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. 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

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

Reviewer #1: Yes

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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

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5. 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

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6. 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: (No Response)

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

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Reviewer #1: Yes: Martin Raden

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