Dear Dr. Schur,

Thank you very much for submitting your manuscript "Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly" (PPATHOGENS-D-19-01294) for review by PLOS Pathogens. We apologize for the delay in getting back to you, but in the summer period it was difficult to secure reviewers. The reviewers found that your study provides interesting insight into the assembly of lentivirus particles, and as such they are an important contribution to the field. As you will see, the main concern was expressed by reviewer 1, and has to do with the way particle release is measured. We feel that it is indeed important for the conclusions you reach to have a way of distinguishing between particles that are correctly assembled but have a maturation default, from released particles that are non-infectious because of an aberrant assembly, and therefore only measuring the fraction of released infectious particles is not sufficient to support your conclusions. We therefore expect that you will be able to answer this point appropriately in the revised version. You will also see that reviewer 2 requests an additional control to validate the observed IP6 density, which if you can provide would be nice, but we do not find it essential for the validity of the paper. Of course, we cannot promise publication until we have seen your revised manuscript.

In addition, when you are ready to resubmit, please be prepared to provide the following:

(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

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We are sorry that we cannot be more positive about your manuscript at this stage, but if you have any concerns or questions, please do not hesitate to contact us.

Sincerely,

Félix A. Rey

Associate Editor

PLOS Pathogens

Michael Malim

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

***********************

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 study, the authors examine the role of IP6 in the assembly of equine infectious anemia virus (EIAV) and several other lentiviruses. By using a variety of approaches, they observe that IP6 markedly stimulates the assembly of EIAV and the other lentiviruses. EIAV Gag is observed to assemble in vitro into several morphologically distinct tubes and sphere; interestingly, the Gag lattice in all these structures is in the immature conformation. Both EIAV and HIV-1 maintain similar immature inferfaces, and both lentiviral Gag proteins bind IP6 via conserved Lys residues in the CA C-terminal domain and adjacent spacer peptide.

In general, the study provides some interesting insights into lentiviral assembly and the role of IP6 in assembly. Some of the results might have been predicted based on earlier work with HIV-1, whereas other findings were unexpected. One major issue, related to how particle production is measured in cell-based assays, needs to be addressed. In their previous work, Dick and colleagues (Nature 2018) measured virus particle production by quantifying the amount of infectious virus released into the supernatant. This approach has a serious limitation: it does not distinguish between effects on particle assembly/release and effects on particle maturation/infectivity. Thus it is not clear from the previous work whether KO of IPPK, a key enzyme in the IP6 biosynthetic pathway, impairs particle assembly/release, maturation/infectivity, or both. This information is vital to understanding the role of IP6 in lentivirus replication. In the current study, the authors use the same approach to evaluate the effect of IPPK KO on virus particle production.

Reviewer #2: In this manuscript, Dick et al. characterise the immature Gag lattice of the lentivirus EIAV. They first recombinantly express different Gag constructs and establish the conditions for their assembly. They do this in the presence or absence of IP6, and find that, while IP6 promotes EIAV Gag assembly, the dependence of EIAV on IP6 is lower than that for HIV. They then structurally characterise some of the assembled lattices by cryo-electron tomography and subtomogram averaging, which allows the authors to produce atomic models for different rearrangements of the capsid domains. They use these atomic models for: (1) studying the key interactions to maintain the lattice; (2) comparing the capsid proteins of EIAV and HIV; and (3) producing mutants to confirm some of their findings. Finally, the authors show that other lentiviruses also require IP6.

Overall, this is a very complete piece of work, with a state-of-the-art subtomogram averaging results, complemented by mutagenesis experiments that confirm the authors conclusions.

Reviewer #3: This paper studied the role of IP6 in the immature particle assembly of EIAV through in vitro and in viro assembly assays, cryo-ET reconstruction and molecular modeling methods. The major results and conclusions of the papers are: (1) IP6 promotes in vitro assembly of EIAV Gag proteins into immature particle structures via conserved lysine residues within the CA-CTD and SP. (2) This function of IP6 is conserved for all lentivirus Gag proteins, although at varied magnitudes. (3) EIAV Gag lattice was resolved from in vitro assembled tubular and spherical structures, at sub-4Å resolution using cryo-ET and subtomogram averaging techniques. (4) When comparing that with HIV-1, the molecular contacts involved with CA-CTD organization are conserved, however the CA-NTD arrangement are not conserved.

Comments, concerns or questions:

1) Although stimulated by IP6, in vitro assembly of EIAV Gag can happen without presence of IP6 (line 331-335). The authors think this is because that in EIAV most of the interactions of the six-helix bundle are hydrophobic in nature, which strengthen the six-helix bundle structure. In contrast to HIV-1, “MD simulations the EIAV 6HB was stable both with and without IP6 likely due to additional side chain contacts between helices in the six-helix bundle” (line 404-405). Can you perform a sequence alignment of this region of all lentiviruses studied in the paper and address if this property is unique for EIAV?

2) Line 264-269: EIAV CA was found to have a shorter helix 1, and therefore in EIAV Gag lattice, the putative dimer interface between neighboring Gag hexamers (found in HIV-1 lattice) is missing. According to HIV-1 mature CA lattice, helix 1 is important in intra-hexamer interactions. Could this be one of the reasons that in the in vitro assembly systems, EIAV Gag always form immature lattice, not mature lattice? Is this property unique for EIAV?

3) The authors found that the EIAV CA-NTD organization is not the same as that in HIV-1. Several molecular contacts in HIV-1 Gag lattice are missing in EIAV which include: (a) there is no intra-hexamer interactions among CA-NTD (line 238-239) (b) there is no dimeric inter-hexamer interaction formed by helix 1 (c) there is no strong contact between CA-NTD and CA-CTD (line 263-264). The studied EIAV assemblies are rather small: narrow tube 35nm, wide tube 70nm and spherical particles are 100nm. In general, retroviruses have a larger dimension. Is it possible that these missing contacts actually present in the authentic particles?

4) Line 109-110: The authors suggests that “the retroviral CA-NTD and CA-CTD are independent structural entities that act autonomously in regulating immature virus particle diameter and curvature”. This idea was repeated at line 363-364: “The spatial separation of EIAV CA-NTD and CA-CTD, and the lack of strong interactions between them, allow these two halves of CA to behave almost as independent entities, as suggested previously for other retroviruses”. This statement seems overly strong. In HIV-1, helix 11 and helix 8 interact with MHR and helix 7. This interaction would limit the orientation of NTD relative to CTD and the role of NTD in deciding the curvature of the Gag lattice.

5) Line 292-295: Line 292 lists the SP sequence as (LAKAL) in which the residue 359 is L. However in line 295, the last residue is listed as T395. Please clarify.

6) Supplement figure 4, the electron density looks low resolution. A better figure is desired.

**********

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. Supp Fig. 3. The authors measure the effect of IPPK KO on EIAV and HIV-1 particle production by measuring the infectivity of virus produced from KO cells. As mentioned above, this approach does not differentiate between effects on particle production and effects on infectivity. The authors need to perform virus assembly assays in which levels of HIV-1 and EIAV Gag in the cell and virus fractions are quantified so that virus release efficiency can be determined, a infectivity values need to be normalized based on viral input (measured by RT or capsid protein).

2. P. 16. The authors speculate about the role of IP6, or potentially other positively charged molecules, in EIAV assembly in vivo. They suggest that, in the cells used, EIAV infectivity is significantly lower than that of HIV-1. Again, this issue needs to be addressed by measuring the efficiency of virus particle production and determining the relative infectivity (normalized for levels of RT or CA) of the EIAV stocks. These crucial experiments will resolve this issue.

3. Line 425. The authors discuss how future work will evaluate the effect of IP6 depletion on assembly, budding and infectivity. This work will need to be performed by measuring virus release efficiency and specific particle infectivity as described above.

4. Supp Fig. 7. Have the authors measured the efficiency of virus particle production of the Q350A mutant in IPPK KO cells? This result would be interesting.

Reviewer #2: My only major concern is related to the assignment of the density for IP6. In order to be certain that this density corresponds indeed to IP6, the authors should obtain an immature lattice average without it (i.e. a negative control), for example using the EIAV GAGdeltaMA preparation with 0µM IP. This is specially required as IP6 sits at the symmetry axis, and non-relevant densities can appear there due to symmetrisation.

Reviewer #3: Comments, concerns or questions:

1) Although stimulated by IP6, in vitro assembly of EIAV Gag can happen without presence of IP6 (line 331-335). The authors think this is because that in EIAV most of the interactions of the six-helix bundle are hydrophobic in nature, which strengthen the six-helix bundle structure. In contrast to HIV-1, “MD simulations the EIAV 6HB was stable both with and without IP6 likely due to additional side chain contacts between helices in the six-helix bundle” (line 404-405). Can you perform a sequence alignment of this region of all lentiviruses studied in the paper and address if this property is unique for EIAV?

**********

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: Minor: Fig. 6A. The sequence of SP1 is incorrect, as is the placement of the CA-SP1 cleavage site. The correct sequence is KARVL/AEAMSQ, where / denotes the cleavage site.

Reviewer #2: - Can the authors estimate the occupancy of IP6 in their averages?

- It would be good if the authors could provide a list/table of the conditions attempted to produce a mature EIAV lattice. Related to this, there is a reference missing in line 473

Reviewer #3: Comments, concerns or questions:

2) Line 264-269: EIAV CA was found to have a shorter helix 1, and therefore in EIAV Gag lattice, the putative dimer interface between neighboring Gag hexamers (found in HIV-1 lattice) is missing. According to HIV-1 mature CA lattice, helix 1 is important in intra-hexamer interactions. Could this be one of the reasons that in the in vitro assembly systems, EIAV Gag always form immature lattice, not mature lattice? Is this property unique for EIAV?

3) The authors found that the EIAV CA-NTD organization is not the same as that in HIV-1. Several molecular contacts in HIV-1 Gag lattice are missing in EIAV which include: (a) there is no intra-hexamer interactions among CA-NTD (line 238-239) (b) there is no dimeric inter-hexamer interaction formed by helix 1 (c) there is no strong contact between CA-NTD and CA-CTD (line 263-264). The studied EIAV assemblies are rather small: narrow tube 35nm, wide tube 70nm and spherical particles are 100nm. In general, retroviruses have a larger dimension. Is it possible that these missing contacts actually present in the authentic particles?

4) Line 109-110: The authors suggests that “the retroviral CA-NTD and CA-CTD are independent structural entities that act autonomously in regulating immature virus particle diameter and curvature”. This idea was repeated at line 363-364: “The spatial separation of EIAV CA-NTD and CA-CTD, and the lack of strong interactions between them, allow these two halves of CA to behave almost as independent entities, as suggested previously for other retroviruses”. This statement seems overly strong. In HIV-1, helix 11 and helix 8 interact with MHR and helix 7. This interaction would limit the orientation of NTD relative to CTD and the role of NTD in deciding the curvature of the Gag lattice.

5) Line 292-295: Line 292 lists the SP sequence as (LAKAL) in which the residue 359 is L. However in line 295, the last residue is listed as T395. Please clarify.

6) Supplement figure 4, the electron density looks low resolution. A better figure is desired.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Dear Dr. Schur:

Thank you very much for submitting your revised manuscript "Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly" (PPATHOGENS-D-19-01294R1) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level, and was found to be improved with respect to the original submission. There are two minor issues that we would like you to consider before formal acceptance, as you will see listed below. In addition, when you are ready to resubmit, please be prepared to provide the following:

(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

(2) Two versions of the manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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. 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.

We hope to receive your revised manuscript within 60 days or less. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.

[LINK]

If you have any questions or concerns while you make these revisions, please let us know.

Sincerely,

Félix A. Rey

Associate Editor

PLOS Pathogens

Michael Malim

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

***********************

The revised manuscript by Dick et al is significantly improved with respect to the original version as the authors have now quantified the amount of released EIAV particles from wild type and IPPK KO cells, showing that there is only a minor decrease in particle release which correlates with the drop in infectiousness, contrary to HIV-1 infected cells for which there is apparently no particle released into the supernatant in the IPPK KO cells. This was one of the main issues raised by reviewer 1 of the original version. The authors have also adequately answered the other questions, except for the request by reviewer 2, who asked to provide a control reconstruction of immature assemblies in the absence of IP6. We had editorially considered that answering to such a request was not a requirement for acceptance, given the amount of work involved and that the observed density for IP6 conformed to the expected shape for this molecule, that it was as strong as the surrounding protein density (including in regions where only 2-fold averaging instead of 6-fold was done). The new version is now acceptable for publication, provided that the authors pay attention to two minor issues:

1. In the discussion, I suggest that the authors add a sentence considering a potential basal immature assembly in the absence of IP6, which would be similar for all lentiviruses but that is detected here in the case of EIAV because the cells used are poorly infectable by this virus. As the same cells are much more susceptible to HIV1 infection, the difference in particle release in the case of EIAV is not very different to basal, whereas it would be more than two logs in the case of HIV1. Depending on the infectivity assays and on the reactivity of the antibodies used in the western blot and on the, this basal assembly in the case of HIV-1 could be missed. In other words, it could be that the striking difference in the requirement for IP6 suggested by Fig. S3 is due to the cell line used for the experiment and does not reflect an intrinsic difference between the two viruses.

2. In Figure 1, I suggest that the authors take a look at the “representative images” that they provide in relation with the quantification provided. For instance, panel A suggests that there are predominantly spheres at 0uM IP and at 10 uM IP5, whereas the quantification in panel B indicates that there are mainly tubes under these conditions. Similarly, panel C (or at least, the enlargement shown) suggests that there are essentially tubes at 10 uM IP5, whereas the quantification in panel D indicates more spheres than tubes. This can be confusing to some readers, so if possible, please provide more representative micrographs in each case (or at least, an enlargement that matches the quantification shown immediately below the micrograph)

Dear Dr. Schur,

We are pleased to inform that your manuscript, "Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly", has been editorially accepted for publication at PLOS Pathogens. 

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens. 

Best regards,

Félix A. Rey

Associate Editor

PLOS Pathogens

Michael Malim

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

***********************************************************

The authors have adequately addressed the minor editorial suggestions made in the previous round of revision, and the manuscript is now acceptable for publication. This is a very high quality paper with data obtained using state-of-the-art electron cryo-tomography and subtomogram averaging methodologies that the authors have strongly contributed to develop. It will certainly be of interest the broad readership of PLoS Pathogens.

Reviewer Comments (if any, and for reference):