PPATHOGENS-D-26-01054

Nucleocapsid-like cryoEM structure of influenza B virus

PLOS Pathogens

Dear Dr. Ballandras-Colas,

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Matthias Schnell

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Additional Editor Comments (if provided):

Both reviewers are very positive about the manuscript, and both comment favourably about its contribution to the influenza virus literature. The issues raised by the reviewers can all likely be addressed with changes to the manuscript text. Please address all of the issues raised in any revised manuscript.

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Reviewers' Comments:

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: The manuscript by Thirion et al. describes the biochemical and structural characterization of influenza B virus NP-RNA assemblies reconstituted in vitro using truncated NP constructs lacking the long N-terminal disordered tail. The authors show that these truncated proteins retain RNA-binding activity and assemble into right-handed antiparallel helical RNP-like particles whose cryo-EM structure resembles previously reported influenza A and D virus nucleocapsids.

Overall, the manuscript is well written and the conclusions are generally supported by the data. The work provides a useful extension of recent structural studies of influenza virus RNP organization to influenza B virus. While the conceptual novelty is somewhat limited, the study contributes valuable comparative structural information across influenza virus types.

Reviewer #2: Thirion et al describe the results of their structural analysis of an influenza B virus RNP-like particle formed by the in vitro reconstitution of NP-RNA. Helical RNP-like particles were only formed when the N-terminal 64 amino-acid residues were removed from the construct. Full-length WT NP was unable to form helical RNP-like assemblies, forming smaller particles instead. RNA binding by the WT and truncated NP was evaluated by fluorescence anisotropy measurements, yielding Kd values that were broadly consistent across all constructs, suggesting that removal of the N-terminal sequence did not impact RNA binding. Negative stain EM of the in vitro reconstituted RNP-like particles showed that they were structurally similar to purified IBV RNPs.

Cryo-EM and 3D image reconstruction of the RNP-like particles with local/helical symmetry imposition let to intermediate resolution maps at 4.1 Å and 5.3 Å resolution for the two truncated NP-RNA complexes. Guided by the previous X-ray structures for IBV NP and prior IAV and IDV cryo-EM structures, a model was assembled into the higher resolution map, aided by the AI map-modification tool EM-ready.

The resulting model provides insights into the oligomerisation of NP – showing for example how the oligomerisation domain forms a loop that inserts into the neighbouring NP protomer, and how the RNA is routed.

Overall the work appears to be expertly performed and in spite of the challenging target some nice maps have been achieved in which the secondary structure elements are well-resolved lending confidence to the interpretation. Considerable detail is presented concerning side-chain interactions both at protein-protein interfaces and in interactions with the RNA. Given the modest resolution, these side-chain placements are presumably heavily guided by prior high-resolution X-ray data and by the most plausible chemistry. Some caution is warranted here, especially as the map has been subjected to a machine-learning map modification routine (although I note that the phenix refinement was performed using the unmodified map).

Although the discussion focusses mainly on what can be seen in the data, throughout the text it is repeatedly asserted that the findings indicate that the N-terminal disordered region does not ‘participate in the helical architecture’ – and some statements around this are a little difficult to understand. Briefly the absence of the N-terminal region allowed formation of extended helical RNP-like assemblies, while its presence prevented that. The assertion that this region is not important for defining the architecture of the helical RNP seems unfounded then. The main finding that the B/NP-RNA complex is structurally very similar to the previously studied A and D RNPs is on far more solid ground. The disordered region however may profoundly impact the higher-order assembly. This could only really be resolved by 3D reconstruction of the authentic RNP, which might shed light on how the disordered region is accommodated to enable formation of the helical RNP, something that may only be possible through replication-coupled RNP assembly.

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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: 1. The main structural conclusion that influenza B RNP-like particles adopt a right-handed antiparallel helical organization similar to influenza A and D vRNPs is consistent with recent studies on influenza A and D virus nucleocapsids. However, the discussion should better acknowledge that the major principles of NP-RNA organization and helical assembly were already established in Chenavier et al., Sci Adv 2023; Chenavier et al., NAR 2025; Peng et al., Science 2025.

2. The conclusion that the B/NP N-terminal tail “does not participate to the helical architecture” should be softened. The data convincingly show that truncated proteins can assemble into helical particles and that the tail is not required for RNA binding in vitro or for the formation of RNP-like architecture under the tested conditions. However, it cannot be ruled out that the tail could plays non architectural role in native vRNPs essential to its proper helical assembly. This is especially important given the proposed flexibility and potential regulatory functions discussed later in the manuscript. I suggest rephrasing this conclusion more cautiously throughout the manuscript.

3. The statement that the reconstituted particles “appear identical to vRNPs extracted from viruses” is overstated. The comparison with native B/vRNPs relies mainly on negative-stain EM morphology. While the resemblance is convincing at that level, the difficulties of obtaining high resolution structure from native vRNPS suggest some intrinsic structural differences. I recommend moderating the wording accordingly.

4. The authors appropriately acknowledge that the RNA density is not sufficiently resolved for de novo modelling. However, this limitation should also be extended to the interpretation of the protein model more generally, as the density displays relatively few well-resolved side chains. At this resolution, the map primarily supports rigid-body or flexible fitting rather than detailed atomic interpretation. Consequently, residue-level interactions should be interpreted cautiously. Nevertheless, I think the cryo-EM density remains sufficiently interpretable to support the main conclusions of the manuscript, especially since several of the interactions discussed (for example those described in Fig. 3C) are consistent with previously reported influenza NP crystal structures.

5. The manuscript still lists placeholder accession numbers (“XXX, YYY, ZZZ”) in the Data Availability section and table 1. These must be completed before publication.

Reviewer #2: (No Response)

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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: 1. The authors may consider clarifying why the full-length B/NP fails to assemble into RNP-like filaments in vitro despite native vRNPs obviously containing full-length NP. A short discussion of possible steric or kinetic effects of the disordered tail during in vitro assembly would strengthen the interpretation.

2. Please ensure all supplementary figures and tables are appropriately referenced and described in the main text.

Reviewer #2: Throughout the text the use of B/RNP-like is an adjective without a noun – I suggest B/RNP-like particles, or B/RNP-like complexes.

Line 41. “extended N-terminal tail does not imper RNA binding ability and oligomeric state…” presumably the intention is ‘impair’ however while it doesn’t appear to have a material effect on RNA binding, it certainly does impact the oligomeric state.

Figure 1C – the negative stain EM of the monomeric NP is difficult to interpret – presumably the white arrows indicate a random selection of the particles, but the image is of a field of particles, why use arrows? – are they pointing to a particular subset? The images are not very convincing concerning the homogeneity/monodispersity of the preparation as the particles are so small. Perhaps other methods may be more helpful, such as dynamic light-scattering or mass photometry (if they are available). The gel-filtration shows a reasonably tight-elution profile but would benefit from e.g. silver stained gel to show protein purity.

Line 146 “Inspection of the micrographs confirmed B/NPWT inability to form helical filaments” why confirmed? Was this information already known?

Line 208 “… we observe a density inconsistent attributable to the RNA in both helical strands”. I am not sure what the intention is here – is the density inconsistent with being RNA? Or is the density weak or discontinuous?

Cryo-EM image processing methods. I note that the authors have used a more or less identical approach to determining the structure of the two N-terminally truncated NP-RNA complexes but implemented in Relion for one map and in CryoSPARC for the other. Is there a reason for this? Was the analysis performed by two operators with their own preferences, or did one software yield a better outcome (considering the different size datasets).

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Reviewer #1: Yes: Loïc Carrique

Reviewer #2: No

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Dear Dr Ballandras-Colas,

We are pleased to inform you that your manuscript 'Nucleocapsid-like cryoEM structure of influenza B virus' has been provisionally accepted for publication in PLOS Pathogens.

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Academic Editor

PLOS Pathogens

Matthias Schnell

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Thank-you for addressing all of the comments from the original reviews. I have reviewed the changes and am happy with all of the edits and clarifications made. Thank-you for submitting your work to PLOS Pathogens.

Reviewer Comments (if any, and for reference):