Peer review of: Cryo-EM structures of HKU2 and SADS-CoV spike glycoproteins provide insights into coronavirus (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The manuscript from Yu et al reports the cryo-EM structures of the spike protein ectodomains from the 
bat alphacoronavirus HKU2 and the porcine alphacoronavirus SADS-CoV, which was isolated in 2017 
and was responsible for severe watery diarrhea of suckling piglets with a mortality rate of up to 90%. 
The 3D reconstruction of the HKU2 spike has a claimed resolution of 2.38 A, which would be the 
highest resolution coronavirus spike reported to date. Overall, these spikes have some features that 
are shared with alphacoronavirus spikes, whereas other features are more similar to betacoronavirus 
spikes. The analysis of the NTDs and clustering into two subtypes was interesting and thoughtful. 
 
Overall, the manuscript is fairly well written, but there are some grammatical issues that need to be 
addressed. The results are a very thorough analysis of these two new structures and all other 
coronavirus structures reported to date, but this can be difficult to read at some points due to the 
variety of acronyms and different nomenclature systems used. The manuscript would likely be better 
suited to a more specialized journal with a strong structural biology focus. Given that there are more 
than 10 coronavirus spike structures already reported, the results presented here do not represent a 
substantial advance in the field, despite the very thorough and thoughtful analysis. There is also some 
concern noted below about the claimed 2.38 A resolution. 
 
Major comments 
1. The authors claim that the C3 reconstruction for the HKU2 spike has a resolution of 2.38 A, but in 
the few figures in which the EM density is shown the resolution appears to be much lower. This is 
particularly true for supplementary figure 1. At 2.4 A there should be waters present. Are waters 
observed in the map? If so, were they added to the model? Some of these concerns could be 
alleviated by including PDB validation reports for the coordinates and maps, which are now mandatory 
for submission to numerous journals. 
 
Other comments 
1. Line 132 lists a break as spanning 204-204 
2. line 158, the phrase ‘regarding to the secondary structure feature’ can be deleted 
3. Line 312 and 313, stand should be replaced with strand 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In the manuscript entitled “Cryo-EM structures of HKU2 and SADS-CoV spike glycoproteins and 
insights into coronavirus evolution” Yu and co-workers reveal high-resolution cryo-EM structures of bat 
coronavirus BatCoV-HKU2 and the related swine acute diarrhea coronavirus (SADS-CoV) spike (S) 
proteins. The authors describe important similarities in the structures of BatCoV-HKU2 and SADS-CoV 
S proteins and while more compact, they resemble the overall architecture of known CoV S proteins. 
Having established such overall structural similarities, they performed a detailed comparative analysis 
of each subdomains of the BatCoV-HKU2 S with that of other coronaviruses for which the structures 
have already been described. By comparative analysis of the N-terminal domain (NTD) of S the 
authors show that BatCoV-HKU2 NTD contains a galectin-like β-sandwich fold and is more closely 
related to the NTD1 (previously named domain 0) domain of the alphacoronavirus HCoV-NL63 than its 
second NTD domain called NTD2 (previously named domain A). By taking structural differences into 
account and using comparative analyses with other CoV NTDs, the authors propose to distinguish 
between two subtypes of NTDs for alphacoronaviruses, with BatCoV-HKU2 NTD being categorized 
within NTDs of subtype I along with the NTD1 of HCoV-NL63 and the NTDs of HCoV-229E and NTD2 of 
HCoV-NL63 falling within subtype II. The C-terminal domain (CTD) of BatCoV-HKU2 was analyzed and 
Yu and colleagues show that its core adopts a structural fold that is more closely related to that of 
betacoronavirus CTDs than other alphacoronaviruses. The CTD domain is typically involved in 
proteinaceous receptor binding and for a number of betacoronaviruses the receptor binding motif 
(RBM) is known to be located in an extended loop/insert region between β5 and β6 strands. However, 
In the case of BatCoV-HKU2 that loop is short. Taking structural features into account, notably the 
layers of β-sheets found in CTDs, the authors delineate two major types: the first is named one-layer 
CTD and corresponds to the CTD of BatCoV-HKU2 and CTDs found in betacoronaviruses such as MHV 
while the second type is named two-layer CTD and corresponds to CTDs found for other 
alphacoronaviruses, gamma- and deltacoronaviruses. Comparisons of the SD1 and SD2 regions which 
connect the CTD with the S2 fusion domain of S, showed that BatCoV-HKU2 retained structural 
features found in other betacoronavirus S proteins. Interestingly, when authors compared the packing 
of BatCoV-HKU2 S protomers within a trimer, they found that it adopts an intra-subunit packing where 
the NTD and CTD of one protomer contact each other. This is also found for other alphacoronavirus S 
proteins such as those of HCoV-NL63 and HCoV-229E and contrasts with the cross-subunit packing of 
beta- and gammacoronaviruses. Finally, the S2 fusion domain of BatCoV-HKU2 appears conserved 
structurally overall, however, due to distinct structural features the S2’ cleavage site, located 
immediately upstream of the fusion peptide is masked by the connecting region (CR) helix and loops. 
 
This is an excellent study which describes for the first time the structure of a bat coronavirus spike 
protein, BatCoV-HKU2 as well as the S protein of the related and devastating swine virus SADS-CoV. 
The detailed structural comparisons the authors conducted allowed them to gain broadly significant 
insights into the molecular evolution of these two viruses, as well as for the evolution of coronavirus S 
protein structural features, such as the NTD. This work will certainly be very helpful in understanding 
in more detail the modular nature of the coronavirus spike, and how that fits in with coronavirus 
evolution and interspecies jumping. Below are a few minor points that would help clarify certain points 
in the manuscript. 
 
1. The authors have undertaken the task of determining the structures of two related coronavirus S 
proteins from BatCoV-HKU2 and the swine SADS-CoV. The latter virus is an example of the capacity of 
coronaviruses to jump from one host species to another, particularly originating from a bat host 
reservoir. With the ongoing global pandemic of COVID-19, caused by SARS-CoV-2 which likely had 
origins in a bat species, it would be interesting for authors to put their findings into perspective and 
discuss the implications of their work in this current context. 
 
2. Line 275: what is the basis for qualifying the β2 and β3 strands of SD2 as “primitive”? 
 
3. Lines 422-424: authors state that “The spikes of bat coronavirus HKU2 and porcine coronavirus 
SADS-CoV (1128 and 1130 residues) are the shortest among all current known coronavirus spike 
glycoproteins”. A paper from 2007 (Dong et al., J Virol 2007, DOI: 10.1128/JVI.00299-07) reported 
the detection of a divergent coronavirus in Asian leopard cats from Southern China. The S protein of 
this virus was found to be very short at only 1035 aa, shorter by about 100 aa compared to BatCoV-
HKU2 and SADS-CoV (GenBank accession no. ABQ39958.1). Protein alignment analysis shows that the 
Asian leopard cat S protein is closely related to porcine deltacoronavirus S, but contains a large 125 
aa deletion in the NTD. Could authors comment on this unusual coronavirus S protein? 
 
4. Below are a few typos and errors that have been noted: 
Line 90: “are essential for the cell entry” consider removing “the” in the sentence 
Line 90: “There are also no reports regarding to the”, please remove “to” here 
Line 227: “To be note”, consider rephrasing to “Of note,”. This expression is found in other places in 
the manuscript (lines 369, 400) and should be changed accordingly. 
Line 242: change “stands” with “strands” 
Line 283: “HCoV-226E” should be “HCoV-229E” 
Line 323: replace “form a turning” with “form a turn” 
Line 338: replace “also named as SeACoV” with “also named SeACoV” 
Line 406: replace “structural feathers” with “structural features”