Peer review of: High flavivirus structural plasticity demonstrated by a non-spherical morphological variant (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This manuscript provides EM structural analysis of a different morphology of flaviviruses, a club-
shaped particle, which exists at mosquito and human body temperature. Most prior structural 
studies of flaviviruses have focused on samples held at 4 degrees which do not exhibit these 
structures. Notably, the club-shaped article accounts for half the DENV3 virions at relevant body 
temperature. 

The club-shaped morphology likely presents antibody epitopes differently which will affect antibody 
efficacy. Prior studies in the field have demonstrated for example, neutralizing antibodies against 
quaternary epitopes and enhancing antibodies against other sites involving the fusion loop. Both 
could be affected by the E protein arrangement in the mosquito and human body-temperature 
morphology. For example, the fusion loop is hidden in the 4 degree structure but appears exposed 
in the body temperature structure. Fab 8A1 binds only to the head and tail, meaning its epitope is 
in a different structure in the club-shaped body. In contrast, Fab C10 binds throughout the particle 
(also indicating that the novel morphology is formed of E like the 4 degree spherical morphology). 

Provision and characterization of this new model is an important advance for the flavivirus 
community and examines a new-to-us but physiologically relevant form of the virion that one may 
see in the mosquito vector and the human host. Antibody binding studies demonstrate that these 
particles are adopted by mature, rather than immature viruses. Other dengue viruses examined 
also show the presence of club-shaped particles as well suggesting this is a broadly applicable 
phenomenon at temperature relevant for infection, although in this study, the DENV3 has a 
greater propensity for club-shaped particle formation than other types of DENV. High fever 
temperature (40 degrees) seems to produce more club-shaped articles from DENV2. Interestingly, 
the structure adopted is irreversible (does not revert to spherical if temp. is dropped). Both club-
shaped and spherical viruses attach to cells equally well. 

EM structures were determined for a club-shaped particle of DENV3 and one from ZIKV and found 
to have different rotations (24 vs 102 degrees) and different axial rise. This leads to a different 
arrangement of protomers of E relative to each other in the rafts. Do the DENV3 particles always 
have thinner shaft while ZIKV broader? 

These results here are a signficiant advance for the flavivirus field as they demonstrate an 
alternate arrangement of E at relevant vector, heathy body and feverish body temperatures that 
must be considered when interpreting antibody elicitation and antibody reactivity, as well as 
vaccine particle production. This paper is provides a key new model and a key new direction for 
the field. 

The writing and figure presentation could be a bit clearer as it was a bit difficult to wade through. 
Editing would better highlight the advance provided by this study. 

Line 83, add “in this morphology” to read “we solved cryoEM helical structures of an antibody-virus 
complex for both DENV and ZIKV in this morphology, and reveal the architecture…” 

Line 95, Why does high osmolarity preferentially lyse club-shaped particles? 
Line 115, Are CH53489 and 8A1 neutralizing? 
Line 166, sentence split in two 

Figure 1c, add labels to left and right panels of 4° and 37° 
Figure 1d, add labels of DENV and ZIKV 
Figure 1e, label head and tail- are these class averages or the individual particle in the red box? 
Figure 2, enlarge +/- RNAse key 

I’m confused the experiment in Figure 2a. Were the viruses transferred from 4 to the indicated 
temps, then lysed vs lysed at 4 then transferred to the indicated temperatures? Please clarify the 
Figure legend and label the experimental panels better. 

Figure 3, label ZIKV on panels a-c and DENV3 on d-e 

Figure 4 is fascinating. Would it be possible to add another panel with circles outlining the epitopes 
of one or known quaternary neutralizing and enhancing antibodies? 

EO Saphire 

Reviewer #2 (Remarks to the Author): 

The manuscript by Morrone et.al., describes a new morphological variant of Dengue and Zika virus 
which is markedly different from the accepted icosahedral structure of these viruses. The paper 
identifies and characterizes the possible structure of this morphology using Fabs against the E 
protein. The manuscript overall reports on an interesting morphology of flaviviruses that would 
have an impact on how we envision the virus infectivity and immune response, but needs more 
explanations. Some comments and suggestions for the authors are below: 

1. How old were the virus samples after purification when conducting these experiments? 
Flaviviruses and other membrane enveloped viruses have been known to show changes in their 
morphology and heterogeneity with time after purification. Figure 1a showing the virus prep at 4C 
already shows many broken and damaged particles, prompting the question as to the quality of 
the virus prep during experiments. 

2. Line 93-96: The authors suggest that the absence of clubSPs on purification from mosquito cells 
at 29C could be due to the clubSPs being lysed in the high osmolarity of purification steps. But 
they have no evidence to support this claim. There could be other cellular factors or serum 
components during virus preparation that prevents the virus from forming clubSPs. This statement 
should be modified to include other possibilities or removed as it implies that the clubSPs are more 
fragile than regular virus particles, for which there is no evidence provided. 

3. Line 100-101: How was the percentage of population of clubSPs calculated? Was it calculated by 
counting particles from cryo-EM micrographs? If yes, then what was the sample size from which 
the numbers were calculated? These need to be explained in the methods to prove the significance 
of the values. 

4. Line 101: The text reads Supplemental fig 1b whereas the actual data is part of Main figure 1b. 

5. DENV2-PVP94/07 strain does not show much clubSP formation, but rather obvious breakdown 
and clumping at higher temperatures. DENV1-WestPac/s clubSPs have markedly different 
elongated structure than that of DENV-3. What happens when the authors incubate the virus for 
longer than 30 minutes at temperatures above 29C? Could this tubular morphology of virus 
particles be a structural stage before the virus loses structural integrity at higher temperatures? 
For example, does the virus go from smooth to bumpy and from there to this tubular structure? 
6. In Fig.1c showing binding of Fab 8A1 to DENV post incubation at 37C, the increased spikiness of 
the head and tail of the clubSPs presumably due to Fab binding is hard to see in the image. 
Addition of a zoomed-in inset comparing it with the uncomplexed control would highlight the point 
effectively. In the current images, it is hard to accept the claim. 
7. Line 124: In continuation to the above comment, the authors claim that the DIII domain of E 
protein is more exposed in the head and tip of tail of ClubSPs, which allows Fab 8A1 to bind. But 
how are they sure that all parts of the extended membraneous structure is covered with protein? 

What if they are uncovered patches of exposed membrane in these tubular virions? How do the 
authors account for it? Even in the case of Fab C10, where the Fab binding throughout the clubSP 
particles is obvious, the images have been acquired by heating the virus post Fab binding, which 
would have influenced the arrangement of the E proteins as discussed in the paper. Do the authors 
have any evidence that the uncomplexed, native clubSPs have a continuous protein cover? It is 
hard to imagine it given the varied dimensions of the clubSPs. 
8. Line127-130: The authors claim that antibody 4G2 binds the clubSPs based on increased 
spikiness of the particles in the micrographs, but again this is very subjective. At the current zoom 
of the images, it is borderline to claim that 4G2 binds the clubSPs. At the minimum, zoomed insets 
comparing the uncomplexed and complexed particles are needed. 
9. Why treat head of DENV as cylinder and not sphere? Is the cylindrical nature of the head 
obvious? Or is it just to minimize the volume calculations? 
10. Line 300-302 in Discussion: The authors state in the manuscript that the Fab was added to the 
virus and then the complex heated for 30min at higher temperatures to form the Fab bound 
clubSP and catSP structures, which were then used for reconstruction. In this scenario, the 
statement in line 300-302 cannot be stated or is irrelevant as when the Fab binds to the virus in 
the experiment, the virus is in its spherical, icosahedral form. Thus, the E protein are in the 
dimeric raft formation already.