Full text loading...
Chapter 13 : Supramolecular Architecture of the Coronavirus Particle
Category: Viruses and Viral Pathogenesis
Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase
This chapter presents the investigation of the supramolecular design of the virion, in the context of a molecular understanding of its component parts. This investigation will illuminate the machinations of viral assembly. The analysis presented here hints at the exquisite interplay of interactions that contribute both form and transience to the coronavirus particle. Coronaviruses can be recognized by their eponymous coronal fringe of protruding spike glycoproteins (S proteins). The viral ribonucleoprotein (RNP) core is populated by the single-stranded RNA genome and molecules of nucleocapsid protein (N protein). The major protein species present in the viral membrane is the triple-pass membrane glycoprotein (M protein), which is central to the virus assembly process. Three coronaviruses deriving from two of the three coronavirus phylogenetic divisions were analyzed in detail using cryo-electron microscopy (cryo-EM) and single-particle image analysis techniques: severe acute respiratory syndrome coronavirus (SARS-CoV), feline coronavirus (FCoV), and murine hepatitis virus (MHV). The analysis of coronavirus particles is aided by their relative structural simplicity: only three conserved high-copy-number structural proteins have been described, and each has distinct biophysical properties that can be used to further assist in identification. Analysis of the radial distribution of density in coronavirus particles revealed a characteristic signature, with external spikes increasingly visible at higher defocus (as in SARS-CoV images), a thin M protein-related density directly apposed to the lipid bilayer, and a somewhat heterogeneous RNP-related feature distributed in the core region.
Key Concept Ranking
- Severe Acute Respiratory Syndrome
Cryo-EM of coronaviruses in vitreous ice. SARS-CoV-Tor2 (A and B), FCoV-Black (C), MHV-OBLV60 (D), and TUN-grown MHV-OBLV60 (E) are shown in “reversed” contrast with density in white. Images were recorded at either ~2.5 μm below true focus (B to E) or ~4.0 μm under focus (A).
Transmission EM comparison of native and spike-depleted coronavirus. Purified MHV-OBLV60 (A) and TUN-grown MHV-OBLV60 (B) were stained with uranyl acetate prior to imaging in order to enhance contrast.
Pleomorphic particles in a typical preparation of SARS-CoV. Average particle diameter, reflecting the mean of the longest and shortest particle diameters (inset), was calculated from cryo-EM images. The cryo-EM image shown here depicts SARS-CoV.
Stratification of density near the viral membrane. Rotationally averaged radial-density profiles were generated for ~30° wedges taken from intact coronavirus particles. Wedges from SARS-CoV (n = 80), FCoV (n = 41), MHV (n = 53), and TUN-MHV (n = 82) particles were aligned on the minimum density node between the headgroup densities of the lipid bilayer. Radial-density plots demonstrate typical interparticle variability in SARS-CoV (left) and an averaged density from different coronaviruses (right). The schematic at the top interprets densities in the spike, membrane-proximal and M protein, and RNP regions.
Analysis of the structural proteins as seen in edge views. Boxed images centered on the viral membrane below one spike were subjected to iterative reference-free alignment and averaging to produce class average images representative of hundreds to thousands of individual images. Edge view class averages show the ultrastructure of the membrane-associated structural protein complex from SARS-CoV (A), FCoV (B), MHV (C), and spike-depleted TUN-treated MHV (D). Intramembrane densities ascribed to SARS-CoV (E) and TUN-treated MHV (F) M protein are indicated with black arrowheads positioned outside each particle. Connecting densities located between the RNP and membrane regions are indicated with white arrowheads.
Analysis of structural protein organization from axial views. Axial spike images were selected from the central region of each virion. (A) Axial images of SARS-CoV (column 1), FCoV (column 2), MHV (column 3), and TUN-treated MHV (column 4), were aligned and averaged iteratively until a stable averaged image emerged (row b). Axial images were filtered in Fourier space to remove image data greater than (row a) or smaller than (row c) 9 nm. Filtered axial images were averaged; the averaged image was refined by 10 rounds of iterative alignment and averaging, and then unfiltered images were aligned to the averaged filtered image for a further two cycles to produce the images shown (rows a and c). Insets show FTs of the corresponding averaged images. The SARS-CoV RNP lattice was used as a reference for iterative alignment and averaging (B). Reflections were selected from the FT of this image (inset) and back-transformed to reveal the overlapping RNP and spike lattices, which are illustrated schematically in panel C. PCA reconstruction was used to clarify spike images from axial views (D). An example eigenimage from PCA of TUN-treated MHV, showing only RNP densities, is presented for comparison (E).
FT analysis of SARS-CoV virion components. One hundred entire SARS-CoV virions, adjacent regions of background vitrified ice, phospholipid membranes, and images of released RNP from spontaneously disrupted particles were selected for analysis. Results are presented as reciprocal space power spectra, showing the intensity of the FT as a function of spatial frequency. Prominent features are noted in the ~15-nm–1 (spike), ~5- to 8-nm–1 (RNP), and ~4- to 6-nm–1 (membrane) frequency ranges.
Description of the structural module present at the coronavirus membrane. Conserved structural proteins are drawn as they appear in axial views (A and B) and edge views (C and D). Images were either compiled from traced densities in class averages (A and C) or composed according to experimentally determined specifications (B and D). Trimeric spikes (shaded midtones) can be seen projecting outward from the membrane, M proteins (solid black) appear as membrane striations, and oval RNP densities are shown in the form of an interior scaffold (lightly shaded). The dimensions of lattices of S trimers ( a = 14.0 nm, b = 15.0 nm, and γ = 100°) and RNP molecules (c = 6.0 nm, d = 7.5 nm, and ε = 100°) were determined from the reflections shown in Fig. 6 B and were consistent with real-space measurements of the same parameters. All components are drawn to the scale shown in panel A.
Scatterplot relating diameter and size for coronavirus particles and empty vesicles. Shown are results for combined SARS-CoV, FCoV, and MHV particles (left; n = 500 total) and, for comparison, vesicles of similar size that were present in coronavirus cryo-EM images but that lacked any visible RNP or spike content (right; n = 23). Diameter refers to the mean diameter for oblong particles, and ellipticity refers to the difference between maximum and minimum observed diameters, expressed as a percentage of the maximum diameter.