Structural Basis for the Immunogenicity of the C-Terminus of VP1 of Echovirus 3 Revealed by the Binding of a Neutralizing Antibody

Round 1

Reviewer 1 Report

The authors present virus-antibody complexes for Echovirus3 and a novel antibody that traps the C-terminus of VP1. They describe the structure and present the potential use of the procapsid in vaccine production.

I have no major issues with this brief communication.

These minor points can be addressed to improve readability and clarity.

line 44 - the authors state that the canyon is the binding site.  Is this proven?  If so, provide the reference.

Line 46 - "... of E3 comprises a single..." (no "of" after comprises)

line 71 - maybe redraft to "... then lysed with 3 freeze-thaw cycles to..."

line 73 - perhaps "... to remove cell debris"  ?

line 81 - perhaps "... with an amount of 50 ug/100uL per mouse"

line 114 - perhaps "... were treated with 6D10 Fab...."

Line 121 - an Arctica microscope cannot operate at 300kV. Do you mean 200kV?

Line 134 - BLOCK-based reconstruction is not fully described or referenced. Is this just a classification of symmetry expanded particles? Or is there more to it?  Please elaborate, or reference the article.

Line 140 - Did you use the "gold standard" procedure of separate half-sets? If so, mention this.

You don't mention sequencing of your antibody.  This should be in the methods...

Line 151 - you don't describe the methods used for SPR. Please include these.

Line 157 - Ahh, here's the Block-reference.  Please add it above.

Lines 166 ff - This is confusing... You say the A-particle is expanded compared with F.  Then you say the E particle is expanded compared to F, but then you say "resulting in E- 169 particles of the same size as F- and A-particles "  Are they the same size or not?

Fig1 - F-particle seems to have the antibody chopped.  You should show a reconstruction where it is entirely visible.

Fig 1G - although colourful, the images presented in this panel are not very informative.  you already show the different conformations of the C-term in panel H.

Fig S3-B - the colour legend is confusing here.  You would be better off just overlaying the footprint of each with some opacity setting. The rimming colours are nearly impossible to make out.  Also, it looks like there is nearly no overlap between 6D10, CD55 and FcRn.  You should point this out in the text.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

In the present manuscript Qi and colleagues use cryo-electron microscopy to obtain a detailed structural analysis of the interaction of the enterovirus Echovirus 3 (E3) with a potent neutralizing antibody (6D10). The authors have obtained high resolution cryoEM maps with 6D10 complexed with all the capsid conformers usually found in enteroviruses: the F-, A-, and E- capsid. Interestingly, employing a careful localized classification method, they show that when 6D10 binds to the E-capsid two distinctive conformations occur named E-upright and E-sideling. The differences are explained by the C-term of VP1 which adopt different conformation in the four different maps reported.

The structural work is of very high quality and I consider the study to be important for virologists and I strongly recommend its publication.

However, while the results are very interesting, in my opinion the authors should address few points before the publication.

1.     The text has to be edited for clarity and for minor typos.

2.     There is absolutely no information in the Methods section regarding the results presented in Fig 1C, D (the SPR experiments).

3.     I think that the current convention for enterovirus terminology is EV-A, B,…  (EV-A71 instead E71).

4.     I suggest splitting Fig 1 into two figures. This way, some details (i.e. 1F) would be more visible. In general, I had difficulties to understand each panel both due to the size and also for the cryptic legend.

Author Response

Please see the attachment.

Author Response File: Author Response.docx