Multiscale Electron Microscopy for the Study of Viral Replication Organelles

Figures

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  Figure 1. Characteristics of different three-dimensional (3D)-electron microscopy (EM) techniques Figure 3. D imaging of cells at different scales and resolutions: (left) volume scanning electron microscopy (SEM) for the imaging of large samples like whole cells and tissue, (middle) electron tomography (ET) of resin-embedded samples to analyze subcellular ultrastructure of specific areas in the cell, and (right) cryotomography to image the biological content in a near-native state. Volume SEM and ET images of resin-embedded samples have high contrast due to the heavy-metal stain used in sample preparation that accumulates on membranes and large macromolecular features. In cryotomography the contrast is much lower as it is only generated by the biological material itself, which mainly consists of low atomic number elements. However, cryotomography allows the direct visualization of macromolecular complexes like ribosomes and polysomes, clathrin coats, ATP synthases, and individual tubulin subunits in microtubules (red arrowheads). Volume SEM and ET can be applied to resin-embedded samples prepared in a similar manner. For transmission EM (TEM)-based methods (i.e., ET and cryotomography),…

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  Figure 2. Invaginated spherules and double-membrane vesicles (DMVs) by electron tomography. +RNA virus-induced replication organelles (ROs) can be divided in two classes—invaginated spherules and elaborate vesicular networks that contain DMVs. (A) Nodavirus- and (B) coronavirus-induced membrane modifications are representatives of these two classes of ROs. (Top) Sketches of the architecture of the invaginated spherules and DMVs induced by these viruses. While the invaginated spherules induced by nodaviruses and other +RNA viruses have neck-like openings that connect their interior with the cytosol, the DMVs induced by coronaviruses appear to define close compartments. (Bottom) Segmented tomographic volumes of the membrane alterations induced by the nodavirus flock house virus (FHV) in Drosophila cells and by the Middle East respiratory syndrome coronavirus (MERS-CoV) in Huh7 cells (adapted from [14] and [39], respectively). FHV induced the formation of invaginated spherules (white) in the outer mitochondrial membranes (blue). Coronaviruses ROs include DMVs (outer and inner membranes in yellow and purple, respectively), convoluted membranes (blue), and double-membrane spherules (orange).…

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  Figure 3. Labeling EM techniques applied to the study of the coronavirus ROs. (A) Immunoelectron microscopy (IEM) detection of dsRNA inside the DMVs induced by SARS-CoV in Vero E6 cells (adapted from [15]). On-section immunogold labeling was performed on sections from plunge-frozen freeze-susbstituted cells using a primary antibody (Ab), a secondary antibody, and protein A conjugated with 10 nm gold. (B) EM autoradiography detection of active viral RNA synthesis showing it is associated to coronaviral DMVs. Tritiated uridine was provided to live Huh7 cells infected with MERS-CoV, so that the radioactive label could be incorporated into newly-synthesized viral RNA. After 30 min, the cells were chemically fixed, prepared for EM, sectioned, and covered by a thin layer of photographic emulsion. In EM autoradiography, the radioactive disintegrations that arise from the sample in random directions create defects in the emulsion that give rise to electron-dense silver grains upon development. The image shows the high density of label in areas containing MERS-CoV-induced DMV (adapted from [39]). Scale bars, 500 nm.

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  Figure 4. Studies of viral ROs in whole cell 3D SEM reconstructions. (A) Focused ion beam SEM (FIB-SEM) segmented volume of a whole human pulmonary epithelial Calu-3 cell at 24 h after infection with SARS-CoV-2 (adapted from [40] with permission). The large-scale data showed how a large network of DMVs (red) and endoplasmic reticulum (ER) (green) spreads throughout the cell, while the resolution was sufficient to resolve small membrane connections between the elements of this network. Other segmented cellular features include Golgi membranes (blue), mitochondria (brown), and nucleus (grey). (B) Whole-cell serial blockface SEM (SBF-SEM) of a CVB3-infected Vero E6 cell at 6 h post-infection. (Top) A slice through the volume displaying abundant ROs in the perinuclear area of the cell. Individual membrane bilayers could not be resolved in the images due to the limited SEM lateral resolution. Despite this, single-membrane, double-membrane and multilamellar ROs could be distinguished by the thickness of the stained membranes (inset: white, hatched and black arrowheads, respectively). (Bottom) Corresponding segmented volume exposing the heterogenous distribution of differently sized RO clusters…

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  Figure 5. Cryotomography of viral ROs. Different possible workflows are exemplified with the recent cryotomography studies of the ROs induced by (top) nodaviruses (FHV) [70,71] and (bottom) coronaviruses (MHV) [72]. (Left) Cryotomography of subcellular structures can be performed on (top) plunge-frozen structures extracted from the cell or (bottom) on intact cells. The latter approach, known as in situ cryotomography or cellular cryotomography, requires the thinning of frozen cells, which can be carried out with a focused ion beam that mills away parts of the cell to create a thin cryo-lamella. Cryotomography revealed the presence of crown-like protein complexes in the necks of nodavirus-induced invaginated spherules (blue boxes) and spanning the two membranes of coronavirus-induced DMVs (orange boxes) (tomographic slices adapted from [70] and [72], respectively). For sub-tomogram averaging, hundreds to thousands of sub-volumes containing these crown-shaped complexes were extracted from the cryotomograms, aligned in 3D, and averaged. Following this approach, the structure of the FHV spherule crown complex was resolved at 8.5 Å resolution (top, surface rendering of the Electron Microscopy…