PREreview of Virion morphology and on-virus spike protein structures of diverse SARS-CoV-2 variants

The SARS-CoV-2 virus has experienced tremendous selective pressure over the course of the global pandemic with variants of concern emerging that differ in terms of transmissibility, immunogenicity, and other properties. The major goal of this paper is to determine how sequence changes in the major determinant of these properties, the Spike protein, affect structure, abundance, and distribution of the virion. In contrast to most previous studies, which use purified Spike that isn’t anchored to the virus surface (or even a mimic of the virus surface or a biological environment), this study uses electron microscopy/tomography to visualize structures in a more native context. The major potential areas of improvement of the paper are the lack of quantitative comparisons to the “meta ensemble” of structures determined by other methods (single particle EM, X-ray, etc) and the limited discussion of the immune evasion properties of the variant structures with regard to antibody binding footprints. It ends with a potential mechanism for how the conformational equilibrium of certain conformations needed for fusion are favored by specific amino acid changes and argues, quite convincingly, that the insights from the in situ structures determined here are less biased in determining such changes. The manuscript therefore succeeds in its major goals - and points to complex interdependencies of different properties in evolution and not a single conformational coordinate as the result of the selective pressure. 

Major points:

• The paper begins with some variation in Furin cleavage and is mostly concerned with structural analysis, but the link between the biochemical properties and the structural/flexibility parameters uncovered is unclear. Furthermore the acronym FPPR is only defined in the Supplementary Figure 1 and the potential importance of the proximal peptide needs a bit more introduction to enable the reader to follow the arguments in the paper.  

• Figure 2d is very tantalizing and potentially shows a simple property that is changing through time. Given that the caveats of small n are already noted in the manuscript, more speculation about the figure’s interpretation could be interesting. It would be good to elaborate in that discussion beyond: “a comparative statistical analysis between strains was not performed, because we cannot take possible variation between virus preparations into account.“ For example, were there notable differences in the preparation?

• Figure 3 focuses on the trimeric structure and identifying key sites - however, it might be useful for colors to be consistent across figures 3a and 3b . This would make the domains easier to structurally identify, and help the reader to associate domains (cleavage site, NTD, RBD, etc.) with the interpretation of the next couple of figures.

• Figure 4 details the structural comparison among variants at the NTD and RBD. It appears that the authors used the same global structural alignment as in figure 3, which may not be the optimal choice to examine local structural variations because overall domain shifts may interfere with the comparison. In addition, no reference or PDB code was included for the reference structure with linoleic acid.

Minor point:

• We are confused as to whether they use B.1 as WT interchangeably in the manuscript. More precision in word choice would avoid some confusion with the figure 1 legend as comparisons also mention WT. In the legend for figure 1, the stats reference comparisons to WT, but some only have B.1 and not WT labels.

• coulombic potential density (or potential density or density) not electron density

• In figure 2c, consider adding a legend that specifies red is prefusion and black is postfusion.

Competing interests

The author declares that they have no competing interests.