Peer review of: Structures of Echovirus 30 in complex with its receptors inform a rational prediction for (2020)

REVIEWER COMMENTS: 

Reviewer #1 (Remarks to the Author): 

This is a very well written, elegant and an important study on the prevalent enterovirus 
Enterovirus 30, causing difficult outbreaks and infections worldwide. There is still very limited 
information on the virus host cell interaction concerning E30 although, recently, Fc receptor was 
shown in two distinguished articles (groups of Coyne and Gao) to act as an essential receptor for 
E30. In this paper, E30 structure was studied in detail and revealed structural changes upon 
receptor binding. Importantly, the authors show that Fc receptor binding causes a collapse of the 
hydrophobic pocket indicative of uncoating already at neutral conditions, while DAF does not 
induce the same changes. The observed structural comparison of full and empty viruses, and the 
neutralizing antibodies elicited even by the empty viruses give hope of their use for effective and 
safe vaccine production. The structure-based in-silico algorithm to evaluate the receptor binding 
area is a novel tool contributing to evaluation of receptor usage of different enteroviruses. 

Major point: 
If I understood correctly, the authors mixed the E30 preparation that had full (50%), empty (43%) 
and A-particles (7%) with the receptor preparations. Why didn´t they perform the binding 
experiments with the purified full, 143S particles from the gradient and not with a mixture of 
structures? How to distinguish the Fc receptor bound collapsed virions from naturally empty virions 
with Fc receptor possibly bound? Did the authors find binding of the receptor to the A particle or to 
the empty virions? 

Minor points: 
The authors could describe in more detail whether the naturally empty virus was without the 
pocket factor? The great stability of the empty virions would suggest that the pocket factor could 
be still inside to confer stability. 

Do the authors think that using the in-silico tool, it is possible to foresee epitopes or groups of 
epitopes which could help in developing better and broadly acting vaccines. Perhaps combining the 
most relevant receptor binding information helps to choose the most important serotypes for 
future multivalent vaccines? 

Do the authors think that the naturally produced empty viruses are a valid way to produce 
vaccines? Do the authors think that E30 VLPs produced in cell cultures would show similar stability 
as the naturally produced empty viruses? 

The developed in-silico method is based on the present and future high-resolution structures which 
allow evaluation of receptor binding sites. Don´t the authors think that the future serotypes that 
are formed by recombination still create a need for making further high-resolution structures in 
order to be evaluated for receptor usage and combating the future outbreaks? 

Methods: 
For how long and at what temperature and buffer conditions were the viruses allowed to bind to 
the receptor preparations before preparing the grids for cryo EM? This information should be added 
to the material and methods. 

Varpu Marjomäki 

Reviewer #2 (Remarks to the Author): 

In the present manuscript by Wang and collaborators ilustrate via a series of high resolution cryo-
EM structures the mechanism by which echovirus 30 (E30) binds its receptors. In the first part, the 
authors describe the cell entry intermediates of E30: the procapsid (E), the full capsid (F) and the 
altered particle (A). This sequence of conformations is well known for many members of 
picornavirus family and places E30 in a wider structural context. Following this characterization, 
the authors determine the structure of the F-particle bound the CD55 and to the FcRn receptors. 
These two structures correspond to two distinct stages in the cell entry process: the attachment of 
the capsid to the cell and the priming the capsid for genome uncoating by releasing the pocket 
factor, respectively. While some of these mechanisms were described for other echoviruses, the 
current study offers a suite of structures that builds a comprehensive view of the molecular details 
involved in each of these steps. 
However, the strongest point of this article is the last part, where the authors propose a structure-
based in silico algorithm capable to predict receptor usage for enteroviruses. This part alone will 
guarantee a wide audience in the field of structural virology and strongly recommends the 
manuscript for publication in Nature Communication. 

Minor points: A series of mistakes, typos and ambiguities must be corrected before publication. 

Line 146: This type of details should be in the Methods section. 
Line 158: The three types of particle (should be particles) 
Line 179: αA helices - Get rid of A 
Line 183: Structural basis for distinguishing the groups within the EVs 
Line 242: Expand the abbreviation SCR = short consensus repeat. It’s not mentioned anywhere. 
Line 338-339: 1,500xg should be 1,500 x g and 4°C should be 4 °C. 120,000xg should be 120,000 
x g. 
Line 341: Same as above. Be consistent in the use hours or h. 
Line 348: 120-kV TEM – provide microscope name 
Line 352: expand the FCGRT abbreviation= Fc Fragment Of IgG Receptor And Transporter 
Line 358: This part should be written with more details. How was the sample prepared: receptor 
mix? Incubation time and temperature? 
Line 364: close should be dose 
Line 374: Starting reference? Or was it ab initio 
Line 379: What Relion version? 
Line 388: was corrected should be were corrected 
Line 416: Particle stability thermal shift assay 
Line 801: kD should be kDa. In the text should add a gap between the number and °C 
Line 846: In figure S2 panel d the authors should use a colour scheme varying with depth that 
makes more clear the features of the maps. 

Mihnea Bostina 

Reviewer #3 (Remarks to the Author): 

The manuscript describes cryo-EM structures of Echovirus 30 (E30) in various states and E30 in 
complex with its attachment or uncoating receptor. Through structural comparisons with other 
EVs, the authors reveal serotype-specific epitopes and specific differences between the subgroups. 
They also deduce the molecular basis for E30 receptor recognition. Besides, they develop a 
structure-based in silico method for prediction of EVs’ receptor usage. Overall, the structural and 
computational studies are coherent, and the conclusion is interesting and may bring new way for 
rational predictions for enterovirus receptor usage. I therefore believe this work is worthy of 
publication in the prestigious Nature Communications. Still, there are a few comments and 

questions the authors need to address. 
1. The authors are suggested to include a brief introduction on the current status of structural 
studies on EVs and EV-receptors, and to make it more clear on the novelty of their current study 
on the EV30 and EV30-receptor. 
2. The current version is lack of cryo-EM data processing details. Please add a supplementary 
figure to illustrate the processing procedure and modify related Methods accordingly. 
3. For the E30-receptor maps, the authors are suggested to show the overall map-model fitting for 
one asymmetric unit, and a zoom-in view on the interaction interface regions. 
4. In P.6 line 187-189, the authors describe that "E30 possesses five additional continuous and 
“star-shaped” protrusions surrounding the mesa, characterized as specific for EV-As, although the 
overall structure of E30 most closely resembles those of other EV-Bs (Fig. 2a)." It is hard to 
recognize the mentioned feature in this figure. Please clarify where this feature locates and how it 
appears on the figure. 
5. In the main text, the authors describe that “we determined the cryo-EM structures of E30 F-
particle in complex with FcRn and CD55”. However, in Table S1, it is listed that “Empty particle in 
complex with FcRn” and “Empty particle in complex with CD55”. Please clarify which kind of E30 
particles was used to obtain these two E30-receptor maps. 
Minor points: 
1) Fig. 3C is too busy, and need to be regenerated to better illustrate the location of the 
interactions and involved residues. 
2. Table S2, third line in this table, “βC” should be “BC”. Also, for Tables S2 and S3, please 
describe how the list of the interactions is generated. 
3. In P.12, line 375, the authors describe “E30-FcRn-complex and E30-4B10-complex”. Please 
clarify where this 4B10 come from. 
4. In P. 11, line 348, please describe the machine type of the “120 kV-TEM” used.