Nature: Reveals the structure and distribution of the new coronavirus hedgehog protein on complete virus particles.

During infection, coronavirus extensively reshapes the internal membrane structure of cells, producing virus replication cytocytostes for viral replication.
S protein, along with proteins M and E, is inserted into the membrane of the endosinosome mesh (ER) and transported to the endosinos-Golgi intermediate compartment (ERGIC).
encapsulated genome germinates into ERGIC to form viral particles, which are then transported to the mass membrane and released.
S protein is prepared for membrane fusion by protease cutting at the S1/S2 bit and then at the S2' bit.
pre-fusion structure of the S-protein fusion of coronavirus, including SARS-CoV-2, has been widely studied through the expression of S-protein isosides in the form of soluble secretion, followed by purification and cryo-EM imaging.
in the pre-fusion structure of the S protein, the receiver binding domain (RBD) is located at the top of a wider S trimer burst structure above the fusion core.
in an S trimer containing three RBDs, each RBD is surrounded by an N-side domain (NTD) that shows a certain amount of fluidity.
In a closed pre-fusion structure, all three RBDs are tiled on the surface of the hedgehog structure, largely enclosing the subject binding bits, while in the open pre-fusion structure, one or more RBDs are raised upward, exposing the binding bits of the binders.
extensive glycosylation occurs on the surface of the S trimer, with 22 potential N-glycosylation points per S protein monomer.
after binding the subject ACE2, the structural transformation from pre-fusion to fusion allows the fusion peptides and trans-membrane domains of the S protein to convergate at one end of a long needle-like structure centered on a three-helix beam.
of the five N-connected polysaccharies along the length interval of the fused S tripolymer prick structure.
fully understand how S proteins work and how they interact with the immune system, it is necessary to understand the structure, composition and distribution of S trimers in viral particles.
a new study, researchers from the British Medical Research Council's Molecular Biology Laboratory and the University of Heidelberg in Germany used cryo-EM to study the structure, composition and distribution of S-thyres on the surface of viral particles.
results were published online August 17, 2020 in the journal Nature under the title "Structures and Distributions of SARS-CoV-2 Spikes proteins on intact virions".
Figure 1. SARS-CoV-2 virus produces features and images from Nature, 2020, doi:10.1038/s41586-020-2665-2.
to avoid artifacts associated with viral enrichment or purification, the authors wanted to image SARS-CoV-2 viruses from the semen of infected cells without concentrating or purifying the virus.
infected VeroE6 cells with SARS-CoV-2 (virus isolated strain Germany/BavPat1/2020).
48 hours after infection, the liquid is clarified and inactivates with formaldehyde and stored at -80 degrees C.
the Western blot, about 45% of the S protein monosomes on viral particles are cut into S1 and S2 at polyserine cutting points (Figure 1a).
the fixed upper liquid is glassed by sudden freezing and imaged via cryo-EM.
may help stabilize some protein building images by crosslinking, but no new ones are expected to be produced.
As expected, taking into account the concentration of viruses in the cellular fluid (approximately 107 phage spot formation units/ml, PFU/ml), they found a small amount of virus particles scattered around the mesh--- these virus particles were imaged through cryo-electronography tom, cryo-ET (Figure 1b).
SARS-CoV-2 virus particles are approximately spherical and have a diameter of 91 to 11 nanometers (n to 179) to the outer edge of the lipid double layer.
they contain particle density corresponding to the N protein and are adorned with S trimers (Figure 1b, c).
these characteristics are essentially consistent with those of other coronavirus using cryo-EM imaging.
There are two forms of S trimers protruding from the surface of the virus -- a few are long, thin structures, reminiscent of the fused structure, and most of them are wider structures, reminiscent of pre-fusion structures.
this observation and a recent paper published on the preprint server (bioRxiv, 2020, doi:10.1101/2020.03.02.972927) --- This paper shows a cryo-EM image of a purified SARS-CoV-2 virus inactivated with the nucleic acid modifier beta-propylene, in which only thin protrusions are observed on the surface of the virus--- in contrast to the virus assembly observed in place.
the authors also collected a layer-scanning map of viral particles produced by SARS-CoV-2 infection with CALU-3 cells, in which the Calu-3 cells are a human lung cancer cell line that produces the same viral titularity as VeroE6 cells.
the morphology and appearance of these viral particles produced by THE CALU-3 cells on the surface of the virus particles is consistent with those observed from the VeroE6 cells.
analysis showed that about 73% of S proteins were lysed.
SARS-CoV-2 virus particles contain 24 to 9 S triple clusters.
this value is lower than the previously assumed iso-distance distribution estimate of the S protein because the S protein is unevenly distributed on the surface of the virus.
small group of virus particles contains only a small number of S trimers, while larger virus particles contain more S trimers.
the authors identified 4,104 wide S trimers and 116 thin S trimers from 179 viral particles and averaged them with sub-tomograms.
structure, at resolutions of 7.7 and 22 E, corresponds very well to the previously published structure of purified S trimers in pre- and post-fusion forms (Figure 2a).
, about 97% of S trimers are pre-fusion and 3% are post-fusion.
S trimer forms before and after fusion appear to be evenly distributed among virus particles.
2. Structural analysis of SARS-CoV-2 S trimers on complete virus particles, pictured is from Nature, 2020, doi:10.1038/s41586-020-2665-2.
pre-fusion S trimer on the surface of the virus may be mainly in a closed configuration, its open configuration can be induced or stabilized by ACE2 binding, or it may also exist before fusion.
when used as immunogens, open or closed configurations induce different ranges of antibodies, and efforts are being made to produce S-protein structures that stabilize one of these building bodies.
to assess the presence of S trimers in open and/or closed configurations, the authors classified the RBD regions of S protein monomers in S trimers.
they found three types: S proteins in closed positions for RBD, S proteins in open positions for RBDs, and S proteins for RBD with closed positions but reduced density, indicating the presence of more m removable configurations.
Taking into account the types to which each S protein monomer is assigned, the authors dedate that the completely enclosed S trimer structure, and one of the RBDs, which is an open S trimer structure, represent approximately 31% and about 55% of the 3,854 pre-fusion S trimers, respectively (Figure 2b).
they also found that two RBDs in a small number of S trimers (about 14 percent of the 3,854 pre-fusion S trimers) were in an open composition (Figure 2b).
these observations confirm that the openness of RBD observed in recombined S trimers also occurs on viral surfaces, and that artificial S protein structures stable in closed and open configurations represent in-place structures.
, this subject binding point is randomly exposed in place and can interact with ACE2 and antibodies.
these S trimers are not protruding directly from the surface of the virus.
they can tilt 90 degrees toward the membrane, although tilting more than 50 degrees is less advantageous.
the authors grouped them according to the direction of the S trimer relative to the membrane, and averaged each group separately.
these averaging structures indicate that the near-end stem area of the membrane is flexible enough as a hinge to allow tilt in all directions (Figure 2c).
the authors built a model of SARS-CoV-2 virus particles in which the position, orientation, and composition of the S triumer are averaging through sub-fault scans (Figure 2d).
S trimer appears to be randomly distributed on the surface of the virus, and there is no apparent clustering or relationship between their position, orientation, and composition.
in SARS-CoV-2, there is about one S trimer per 1,000 square nanofilm surfaces, compared with about one S trimer per 100 square nanofilm surfaces in influenza A viruses.
in SARS-CoV-2, the sparse distribution of S proteins and their largely closed state mean that the binding of the subject may be less dependent on affinity effects than pandemic influenza viruses.
this is consistent with a higher affinity (in the nM range) between S protein and ACE2 than between hemoglobin and salivary acid (mM range).
low concentrations of virus particles in the upper liquid make it difficult to resolve high-resolution structures.
, the authors concentrated these viral particles using centrifugal precipitation of sucrose pads.
concentrated virus particles deviate from the spherical form, but their overall characteristics remain.
they averaged these viral particles with cryo-ET imaging and sub-fault scanning maps, mainly in pre-fusion S trimers, and occasionally post-fusion S trimers.
classifying pre-fusion S proteins, they were able to identify only RBDs in closed positions and observed S protein monosomes with weak RBD density.
the virus particles in the upper liquid from infected cells mainly show pre-fusion S trimers, which are in closed or open pre-fusion configurations.
particles concentrated by centrifugation of sucrose pads continue to present pre-fusion images, but open compositions are no longer observed.
other studies have shown that virus particles inactivated with beta-propylene esters, rather than formaldehyde, are mainly in a post-fusion state.
S trimer purified from the membrane is only in the closed pre-fusion and post-fusion composition, while other studies have suggested that the open RBD in soluble S trimer is found in a series of consecutive different locations.
observations suggest that the open pre-fusion configuration of the S protein observed by the authors before, rather than after, was fragile (although fixed) and could be affected by the purification process.
authors suggest that inactivation and purification methods can change the ratio of pre- and post-fusion forms, as well as those of open and closed forms.
it is speculated that a large amount of fused S protein on the surface of the virus may protect the virus by shielding the pre-fusion form, or may shift the host reaction to non-mediated antibodies.
In view of the observation of a small part of the fused S trimer prick structure on complete virus particles, these authors believe that this is unlikely to be an important defense mechanism for the virus during infection, but it may be an important consideration for vaccination.
vaccine based on inactivated virus particles is under development.
these vaccines may present different S protein tables to the immune system depending on how they are prepared, so their ability to induce and react is different.
For example, beta-propylene ester is often used in vaccine production (e.g. for influenza virus sub-unit vaccines), but if the S trimer induces non-neutroactive reactions after fusion, then beta-propylene ester may not be the best choice for virus inactivation during the preparation of the SARS-CoV-2 virus S protein vaccine formulation.
Next, the authors used cryo-EM to perform two-dimensional imaging of the concentrated SARS-CoV-2 virus and to analyze the pre-fusion S trimers protruding from the side of the virus particles, resulting in a 3.4-E resolution pre-fusion S trimer consensus structure.
the RBD monosomes are centrally classified and partially subtracted, which can be divided into two categories.
consistent with the absence of open compositions in samples found through cryo-ET, the authors observed that 81% of S-protein monosomes had RBDs in closed configurations and 19% of S-protein monosomes had weak density, but were mainly in closed positions.
they optimized the structure of all three RBDs in the closed configuration of the S triumerate (53% of the data) and at least one RBD in the reduced density of the S trimer (47% of the data) to 3.5 and 4.1 E resolutions respectively (Figure 3a).
the two structures are highly similar and differ only at the density level of one RBD.
using this structure with three closed RBDs, they built and perfected an atomic model of the S trimer in place on the surface of the virus.
3. Using a single particle reconstruction method to determine the structure of the SARS-CoV-2 S trimer on complete virus particles, pictured is Nature, 2020, doi:10.1038/s41586-020-2665-2.
in the structure established by these authors.