Dear Dr Pichlmair:

Thank you very much for submitting your manuscript "The alternative cap-binding complex is required for antiviral defense in vivo " (PPATHOGENS-D-19-01246) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved. In particular, reformat the manuscript as suggested to make it more comprehensive, and study the abundances of few more selected ISGs to solidify the data.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

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Sincerely,

Peter Sarnow

Guest Editor

PLOS Pathogens

Sara Cherry

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In this work, the authors look at the role of NCBP3 alternative cap binding complex during viral infection. They show that the loss of this protein leads to a decoupling of transcription and translation of antiviral genes, which increases susceptibility to viral infection. The data are generally of high quality and I think the story is compelling an on an important topic.

Reviewer #2: Coordinated regulation of gene expression is essential for a proper innate immune response in response to infection. This can happen at any stage of gene expression, including transcription, mRNA stability and translation. Previously, Gebhardt et al discovered that an alternative cap-binding complex, consisting of NCBP1 and NCBP3, which is important during cell stress like VSV, EMCV, and SFV virus infection. Following upon this first paper, here Gebhardt et al determine the effect of NCBP3 knockout on Influenza A infection. They generate NCBP3 knockout mice, and show that in knockout cells IAV replicates to a higher titer compared to wild type cells. This increased replication correlates with decreased Ifnb1 mRNA levels, suggesting that NCBP3 is important for the cell to establish an antiviral state. Notably, mRNA levels of another ISG, Ifit3, are unchanged, indicating there is specificity to the regulation by NCBP3. To determine the proteome dependent on NCBP3, Gebhardt et al perform proteomics in IAV-infected WT and NCBP3 knockout cells. They discover that ~5% of the detected proteome is altered upon knockdown, including 19 ISGs. In agreement to the importance of NCBP3 in establishing an antiviral state, cells do not correctly respond to interferon or PIC treatment, and knockout mice have significantly reduced survival rates upon challenge with IAV.

Overall, this is a very nice paper that implicates NCBP3 as an important player in the antiviral response. I have minor specific suggestions and questions about some discrepencies in the data:

1. Fig 1: Where is the western blot for the efficiency of NCBP1 knockdown by siRNA?

2. Fig 4B: Why are peptides for IAV proteins detected in the mock infected samples?

3. Fig 4: Please add gene ontology analysis of all 282 proteins whose expression is changed upon NCBP3 knockdown. Are there other stress response pathways being altered besides the ISG response?

4. Fig 4: In the prior paper discovering NCBP3, Gebhardet et al performed CLIP with NCBP3 – what population of mRNAs that crosslinked to NCBP3 encode proteins that are altered in the proteomics experiment? Does enrichment of RNA binding correlate with the level of change in protein levels upon knockdown?

5. Fig 2/5: In Fig 2, IAV infection induces the same amount of IFIT1 protein in both wild type and mutant cells, suggesting that NCBP3 is not important for the increase in IFIT1 levels upon infection. However, in Fig 5, IFIT1 protein levels are reduced in NCBP3 knockout cells upon IFN treatment. Why is there this discrepancy?

In addition, these data do not agree with the text on page 13/14, which should be reworded: “…expression of ISGs, monitored by Ifit3 mRNA and IFIT1 protein, did not scale with severity.” - in Fig 2C the levels of Ifit3 mRNA are identical between knockout and wild type cells, and the levels of IFIT1 protein also are the same in both cell types. In addition, despite in the SI table, IFIT1 is a “non-sig ISG,” on p14/15, Gebhardt et al speculate that Ifit1 mRNA stability is affected by NCBP3. This section should also be reworded.

Reviewer #3: The submitted manuscript by Gebhardt et al. reports the role of NCBP3, which along with NCBP1 forms an alternative cap-binding complex (CBC), in antiviral defense against RNA viruses in vivo. Through a series of ex vivo-, proteomic-, and in vivo-based approaches, the authors determine that NCBP3 is necessary to mount an antiviral response, and there is a unique subset of ISGs that are significantly regulated by NCBP3. The authors use MEFs from Ncbp3+/+ and Ncbp3-/- mice to demonstrate lack of NCBP3 results in enhanced virus growth and a dampened antiviral response. These data are corroborated by survival studies and histology in vivo.

This manuscript is an extension of the work done by the same group published in Nature Communications in 2015, in which the authors nicely describe an alternative CBC in which NCBP2, the canonical nuclear cap-binding protein can compensate if NCBP3 expression is lost, and that NCBP3 is important under conditions of virus infection. The current manuscript significantly expands on this idea by moving to an in vivo model (newly generated Ncbp3-/- mice ) and by utilizing several different viruses while also investigating in greater detail the antiviral response. Overall, the experiments are well-executed and the data convincing. There are only a few remaining points that the authors should address in order for this manuscript to be considered for publication:

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: My major critique is that the figures appear somewhat disjointed and the message of the report becomes jumbled. As I interpret the data, the key message is that loss of NCBP3 affects translation of certain antiviral genes. The proteomic data shows this at the protein level for a subset of ISGs, and for some genes, the authors show that RNA levels aren’t affected. There however, is no systematic presentation of the data. Sometimes they show RNA of one gene, sometimes protein from another, and in essentially all cases, it is not clear why a specific gene is selected (especially of the IL6 data). Furthermore, none of the validation data appears to be linked to the proteomic data, which is a centerpiece of the paper.

Some specific comments for the figures are below. But overall, I believe the impact of the paper would be significantly strengthened with some reformatting. If the report showed a systematic determination of which antiviral genes had defects specifically in translation after the loss of NCBP3 (maybe RNAseq combined with the proteomic data), and then those genes were validated in vitro, the story would be much more digestible. As currently presented, the genes individually tested before (and after) the proteomics don’t appear to come out as hits, and it is difficult to understand why the specific genes tested with qPCR and western were chosen (or how they relate to the in vivo phenotype). The phenotypes are clear, and the in vivo data are striking, but it is currently difficult to understand the scope and magnitude of the NCBP3 effects on antiviral gene expression.

Major Comments:

Figure 2C: This analysis should have statistical significance indicated, and the authors are concluding sometimes the knockout significantly changes things and sometimes not.

Figure 2C,D: Why was IFIT3 quantified at the RNA level and IFIT1 at the protein level? Do different ISGs all behave the same way? Showing several ISGs at both the RNA and protein level would be informative.

Figures 2,3: The interpretation of antiviral gene expression is somewhat complicated by differences in viral replication. Transfecting PolyIC may be a “cleaner” way to quantify antiviral response in the absence of effects of viral replication. The knockout phenotype may be more apparent in this system.

Figure 4: It would be nice to have matched RNA levels for this experiment, this could support the data from figure 2.

Figure 5: Why was IL6 selected? There is no clear link to IL6 from any of the previous experiments that I could find.

Reviewer #2: Comment 5 from the above suggestions needs to be addressed, specifically the discrepencies between the different data needs to be addressed, or the Ifit mRNAs should not be highlighted in the discussion as NCBP3 targets.

Reviewer #3: Figures 2C/3C. Additional ISG genes should be measured in order to characterize better the IFN/ISG response in wt vs knockout cells. Since the authors describe a subset of differentially regulated ISGs in Figure 4, these would also be nice to test and validate by qRT-PCR.

Figure 5. Similarly to the point above, it would be nice if the authors could test additional genes to make a better generalization of the antiviral activity of NCBP3. Additional chemokines (like CCL5 tested by ELISA), type I IFN (IFNB1) or ISGs (IFIT1, IFIT3, MX1, OAS), and particularly the ones identified by the proteomic analysis, would be nice to show and supplement the current data.

To confirm that NCBP3 is controlling the antiviral effect, Ncbp3-/- MEFs should be reconstituted with NCBP3, followed by virus infection.

Figure 6. What is the effect on viral titers (or viral mRNA) in wt vs knockout mice, e.g. in the lungs? Is there any differential cytokine and ISG regulation in these mice?

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: The light blue and light red colors are sometimes difficult to distinguish, perhaps darker variants of each color would help with clarity.

Reviewer #2: See comments 1-4 in summary.

Reviewer #3: Figure 2C. Neither error bars nor significance are provided for these qRT-PCR data.

Figure 4. Could the authors describe how ISGs were grouped and defined?

Figure 4D. It is unclear to the reviewer what the difference is between the pink (non-significant ISG) and green (significant ISG) as the text states, “Overall, we have identified 282 proteins that change their response to IAV infection upon NCBP3 knockout, including 19 ISGs (Fig 4D, S1 Table).” However, from the figure, there seem to be pink dots overlapping with green ones, which appears that significance would be quite similar.

Figure 6B. Arrowheads could be used to make areas of lymphocyte infiltration clear in the slides.

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Dear Dr Pichlmair,

We are pleased to inform that your manuscript, "The alternative cap-binding complex is required for antiviral defense in vivo", has been editorially accepted for publication at PLOS Pathogens. 

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Best regards,

Peter Sarnow

Guest Editor

PLOS Pathogens

Sara Cherry

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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Reviewer Comments (if any, and for reference):