Dear Dr Pasdeloup:

Thank you very much for submitting your manuscript "Dynamic organization of Herpesvirus glycoproteins on the viral envelope revealed by super-resolution microscopy" (PPATHOGENS-D-19-01587) 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.

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

Sincerely,

Yasuko Mori

Guest Editor

PLOS Pathogens

Erik Flemington

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 manuscript, Beilstein et al investigate the organization of several envelope glycoproteins (gps) of herpes simplex virus-1 (HSV-1) at the surface of cell-free and cell-attached viral particles. They use stimulated emission-depletion (STED) microscopy, a new tool which allows to achieving an average visual resolution of 60 nm, i.e. inferior to the size of viral particles (200nm), to visualize the sub-structural composition of the viral envelope. They chose to investigate 5 of the main envelope gps (gC, gD, gB and the gH-gL complex) implicated in cell entry using a panel of monoclonal and polyclonal antibodies, to explore their respective localization at the envelope surface, their colocalization, and putative redistribution due to cell attachment. To obviate any bias, they carefully compared results obtained with monoclonal and polyclonal antibodies, in particular when they analyzed colocalization of two gps, by combining a polyclonal antibody directed to one gp with a monoclonal antibody directed to the second, in both associations.

The main results suggest that gD localizes differently in cell-free and cell-attached particles; that gps functionally involved in entry, gB and gH-gL, overlap on cell-free particles; that all glycoproteins except gC converge and are dynamically redistributed around cell-bound particles.

The experiments are cautiously done and the results are precisely described and overall convincing, and provide new insights which should be of great interest to the scientific virology community. I however feel that several clarifications could improve the manuscript.

Reviewer #2: Stimulated emission depletion microscopy (STED) is a fluorescence microscopy technique that overcomes the diffraction limited resolution of confocal microscopes. The manuscript by Beilstein et al. explores herpes simplex virus 1 (HSV-1) glycoproteins on the purified virions through this new technique. They found that gB and gH/gL are distributed around purified virions., although the other essential glycoprotein gD localizes as clusters which are distinct from gB and gH/gL. Furthermore, they reported that all glycoproteins showed a similar ring-like pattern when virions were adsorbed to the cells, but the diameter of these rings was significantly smaller than those observed on cell-free viruses. Their results suggested that gD can be reorganized on the viral envelope following either a possible maturation of the viral particle or its adsorption to the cell. The authors suggested the model, in which the redistribution of glycoproteins during adsorption could contribute to initiate the cascade of activations leading to membrane fusion.

The article is well written, and clearly shows the unexpected character of viral glycoproteins during entry using super resolution microscopy. As it is not totally understood how viral glycoproteins on the virions induce membrane fusion during viral entry. New techniques such like super resolution microscopy and live imaging should shed light on viral entry, which is essential for viral replication. Thus, this article will be of broad interest in the readership. I have several suggestions for improving this manuscript.

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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: No supplemental experiment required. The authors should provide several additional statistical analyses (see below).

Reviewer #2: (i) Upon binding to the cells, the authors claimed the rings of glycoproteins were constricted. However, it is many possibilities in addition to the change on the position of glycoproteins. The authors should mention the other possibilities, such as global change of the modifications of epitopes on the glycoproteins.

(ii) Can the authors estimate the number (ratio) of gD among virions?

(iii) The authors should show the methods of collecting the virions. Such as cell lines, MOI or times after infection. These conditions effect composition of virions dramatically.

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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. Have epitopes recognized by the different mAbs been identified, and if so can the authors provide their localization? This information could be helpful for interpretation of results (see lines 170-179, 231, and 347-348).

2. The authors report 4 different profiles of gp localization, but they mainly distinguish the “ring-like” as opposed to the other three profiles in their interpretation of results (Fig 1). First, do the authors suggest that the single, double and multiple profiles correspond to a similar functional state as opposed to the ring-like, or that each of these profiles correspond to different states/ functions? Second, were there non “ring-like” profiles containing more than 3 spots, or is 3 the largest distinguishable number of individualized spots in the multiple profile (i.e., how do the authors differentiate the multiple and “ring-like” profiles)? This point should be clarified since the whole discussion is based on this distinction : in particular, adding the “multiple” and the “ring-like” profiles would eliminate the distinction between the cell-free and the cell-attached localization of gD in HFF cells, and in HeLa cells analyzed with a pAb.

3. The results obtained using polyclonal and monoclonal antibodies are not strictly identical, mABs tending to provide more ring-like images on cell-bound particles than pAbs (see Fig 2 gB, gD). Are these differences significant and if so, how can they be explained? (see Q1).

4. A constriction of the diameter of rings of gB, gH-gL and gD is observed when comparing cell-free and cell-attached virions, ranging from 12 to 21% (Fig 3). There is also a difference in the diameter of gp rings on cell-free virions using pAbs compared with mAbs (Fig 3A, see gB and gH/gL, and Fig3B, see gB and gD): is it significant and how can it be explained?(see Q1).

5. Figure 5 : the interpretation that spots of gD and gC colocalize (lines 243-244, and 250-251) is not obvious (compare lanes x and xi).

6. Figure 6 C : The results show that gD is clustered at the surface of cell-free particles, and redistributed in a ring-like organization in cell-bound particles. As the authors remind (p 14 line 316), glycoproteins involved in cell binding and fusion should regroup locally at the point of contact with cells : indeed maturation of HIV particles is contemporary with the egress of mature particles from cells, and env clustering is supposed to render the virus productive for infection (Chojnacki, Nat Commun 2017). Therefore, the results shown in this manuscript seem to contradict previous results obtained in another model. Can the authors clarify further this point in their discussion ?

7. Since redistribution of gD was observed at 4°C, i.e. in conditions which permit attachment but preclude fusion, can the authors comment on glycoprotein mobility in membranes and in particular at that temperature?

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Reviewer #1: No

Reviewer #2: No

Dear Dr Pasdeloup,

We are pleased to inform that your manuscript, "Dynamic organization of Herpesvirus glycoproteins on the viral envelope revealed by super-resolution microscopy", has been editorially accepted for publication at PLOS Pathogens. 

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Best regards,

Yasuko Mori

Guest Editor

PLOS Pathogens

Erik Flemington

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):