Multiscale Electron Microscopy for the Study of Viral Replication Organelles

Abstract

During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in the cytoplasm of the infected cell. These viral replication organelles (ROs) play a key role in the replicative cycle of important human pathogens like coronaviruses, enteroviruses, or flaviviruses. From their discovery to date, progress in our understanding of viral ROs has closely followed new developments in electron microscopy (EM). This review gives a chronological account of this progress and an introduction to the different EM techniques that enabled it. With an ample repertoire of imaging modalities, EM is nowadays a versatile technique that provides structural and functional information at a wide range of scales. Together with well-established approaches like electron tomography or labeling methods, we examine more recent developments, such as volume scanning electron microscopy (SEM) and in situ cryotomography, which are only beginning to be applied to the study of viral ROs. We also highlight the first cryotomography analyses of viral ROs, which have led to the discovery of macromolecular complexes that may serve as RO channels that control the export of newly-made viral RNA. These studies are key first steps towards elucidating the macromolecular complexity of viral ROs.

Keywords: blockface imaging; coronaviruses; double-membrane vesicles; electron tomography; in situ cryotomography; invaginated spherules; nodaviruses; positive-strand RNA viruses; virus–host interaction; volume SEM.

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), the samples have to be thinned to a thickness of 100–300 nm prior to imaging. SEM- and TEM-based approaches apply different principles to obtain 3D reconstructions of the specimen. In volume SEM, serial 2D images through the sample are combined to obtain a 3D stack. These 2D images are obtained, for example, by repeatedly imaging the sample blockface after removal of a thin top layer in the material. While the lateral resolution is determined by the diameter of the scanning electron beam, the z-resolution is ultimately limited by the thickness of the layers removed. For electron (cryo)tomography, the specimen is gradually tilted in a TEM to generate a so-called tilt series that consists of 2D projection images of the specimen at different angles. These projections are computationally aligned and combined into a tomographic volume. Due to the geometry of the sample holder and of the own sample (cell sections with slab geometry), the tilt angle is restricted to a maximum of ~70°. This incomplete angular coverage creates an effect, called the “missing wedge”, which results in lower resolution in the z-direction [5].

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). These coronavirus-induced membrane structures are often interconnected, either directly or through the ER from which they derive. Scale bars, 100 nm.

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.

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 (multicolored) around the nucleus (blue) and within the cell (beige, semitransparent). Images adapted from [59]. Scale bars, 2 µm.

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 Data Bank (EMDB) entry EMD-22129, [71]), and the structure of the DMV-spanning MHV pore complex was determined at 30.5 Å (bottom, rendered from EMDB entry EMD-11514, [72]).