Peer review of: A glycoprotein B-neutralizing antibody structure at 2.8 Å uncovers a critical domain for (2020)

Reviewer #1 (Remarks to the Author): 

Olivier and colleagues present in this work the structure, by cryo-EM at 2.8Å resolution, of varicella-
zoster virus (VZV) native gB protein in complex with a neutralizing human monoclonal antibody, 93k. 
The glycoprotein gB together with gH-gL forms the viral fusion complex and post-fusion structures of 
gB ectodomain have been solved for several members of the Herpesviridae family to which VZV 
belongs. However, the structure of native pre-fusion gB remains elusive. In this paper, the authors 
isolate native gB from VZV infected cells and use a neutralizing antibody to identify protein domains 
important for viral fusion. Using several mutants, designed based on the structural data, they identify 
domain IV, that is distant from the gB fusion loop located in DI, as a critical region required for 
herpesvirus fusion. 

The paper is well written and the quality of the figures and especially the movies is remarkable. I 
enjoyed reading this work and I have two main points that I feel should be addressed more explicitly 
in the text concerning the conformation of gB and the mechanism of action of the 93k antibody. 
The neutralizing 93k antibody presumably acts by binding to the prefusion form of gB and the 
approach to isolate native gB from VZV-infected cells would suggest the possibility to capture gB in 
the pre-fusion conformation. Why the recovered native protein is in post-fusion conformation? Was 
any pre-fusion/antibody complex recovered? 
What is the hypothesized mechanism of action of 93k? If 93k blocks fusion should not prevent the 
prefusion to postfusion transition? Is it possible that the MOA intervenes at levels other than fusion 
(receptor? binding of other viral proteins?). 

The native full length gB protein has been isolated from the infected cells but the structure reported is 
the one of the ectodomain. Western blot analysis with an antibody recognizing the cytoplasmic region 
of the protein showed that the protein in the complex included the transmembrane domain. Was this 
part of the complex visible in the cryo-EM structure (as it seems from figure 1D)? 

Minor points concerning the figures and the presented results are following: 

Figure 1. This figure is too dense and especially the upper part depicting the functional assays is too 
small. The foci and syncytia are barely visible to be appreciated. The two bar graphs in 1A and 1B 
could be fused showing UT – 93k – 93kF -SG2 – 206 data in one graph for each figure. For figure 1A 
the y axis of the graph should indicate PFU or N. of plaques and not PFU/ml. It would be easier to 
express the values as percent of untreated. I assume that the two bands in the WB of figure 1C are 
the results of the furin cleavage of gB but this is not mentioned in the text. I would add a color legend 
indicating DI – DII etc in figure 1E. I would also remove the bottom part of figure 1F which is well 
detailed in suppl. figure 3 as well as in figure 2. This will allow more space to enlarge the top of the 
figure. 

Figure 4. Figure 4B is too small. I would also show the error bars in black otherwise it seems that the 
top of the empty bars is filled and it may induce some confusion in the understanding of the graph. In 
my opinion, the authors underestimate the impact of the difference in expression (especially at the 
cell surface) of the mutant proteins in the cell-cell fusion assay. It is possible that a defined threshold 
is required for cell-cell fusion to occur so even small variation in protein expression may have a higher 
impact in the fusion read-out. The fusion data for each mutant should be normalized for the amount of 
surface protein expressed. Also, the authors could comment on the different signal with the SG2 and 
93k antibodies, I would have expected some loss of signal with 93k because the mutants are targeting 
the antibody binding site but why some mutants seem to react less with SG2 than 93k? This would 
imply that the binding domain of 93k is overlapping with the one of SG2 (as suggested later in the 
paper) and the mutation is affecting the recognition of this antibody too. In this case to quantify 
expression of the mutants should not another antibody (not affected by the mutations) be used? Could 
the authors comment on the following observations? Figure 4E: The 596-AA-597 mutant does not 
show the cleaved fragments when immunoprecipitated with 93k but these forms are present in the 

immunoprecipitation with SG2. Figure 4F: the western blot for gB shows only one band, is the cleaved 
form not part of the gB/gH-gL complex? 
The control gH lane indicated in the legend (pag.44 line881) is not depicted in the figure. 

Figure 5. The E670A mutant seems to drastically affect fusion both in cell-cell fusion assay and in the 
context of the infectious virus. Why the double mutant (which include this mutation) behaves 
differently in the context of the virus infection? Figure 5F: If the gH-V5 protein was used for the 
coimmunoprecipitation assay, why gH-WT line does not show a band with the V5 antibody? 

Suppl. Figure 2. The naming of the figures is shifted B should be A, C should be B etc. 

Reviewer #2 (Remarks to the Author): 

Members of the Herpesviridae are pathogens for human and animal diseases. The glycoprotein gB 
mediates essential physiological process like viral entry and membrane fusion. However the 
mechanism for gB pre-post fusion transition and the factors triggering membrane fusion are still 
unclear. This study reported a neutralizing human mAb 93k targeting VZV gB domain IV (DIV) and 
found the 93k inhibits membrane fusion mediated by VZV gB. The authors further determined the 
2.8Å cryo-EM structure of native full-length VZV gB trimer in complex with mAb 93k Fab fragments. 
Based on the high resolution cryo-EM structures, the authors further used mutation and functional 
assays to identify critical residues from the 93K epitopes on the DIV for gB-mediated membrane 
fusion initiation. The authors also analyzed these key positions among other Herpesviridae gB 
orthologues by sequence alignment and structural comparison, thereby extending the insights from 
the current VZV study to other herpesviruses. I think this study is of high quality, both in structure 
determination and the subsequent mutation and functional assays. 

Here are my questions and comments: 

1. The Figure 3C clearly shows the interacting residues between the DIV beta stands and the antibody 
93K. Besides the important residues in the beta23 and beta30 revealed as in Figures 4 and 5, the 
authors did not check the residues of other beta stands, especially the beta25. Will these interacting 
residues in other beta stands also have potential influence on fusion? 

2. The authors mentioned other reported DIV antibodies of gB orthologues (HCMV antibody and HSV-1 
SS10) and the SS10 have key residues corresponding beta30 of VZV gB. I advise the authors to show 
their epitope patch in the structure for clear comparison. The SS10-interacting resides in the beta30 of 
HSV-1 gB are also important for fusion of HSV-1? 

3. Will the antibody 93k affect the interaction between VZV gB and heparan sulfate proteoglycans? 

4. There are no control and gB-WT images in Figure 5C and 5D, respectively. 

5. In Figure 4F and 5F, the WB-93 should be WB-93K. 

6. In Figure 2, Figure 3B and Figure 3C, the images of interacting residues are all too crowded. 
Especially in Figure 3C different panels, there is no need to show residues not related to interact with 
specific DIV residues.