Integrative Imaging Reveals SARS-CoV-2-Induced Reshaping of Subcellular Morphologies

Abstract

Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2-infected human lung epithelial cells. We report 3D electron microscopy reconstructions of whole cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodeling of cytoskeleton elements. Pharmacological inhibition of their dynamics suppresses SARS-CoV-2 replication. We thus report insights into virus-induced cytopathic effects and provide alongside a comprehensive publicly available repository of 3D datasets of SARS-CoV-2-infected cells for download and smooth online visualization.

Keywords: FIB-SEM; Golgi; coronavirus; cytoskeleton; electron tomography; intermediate filaments; live cell imaging; membrane remodeling; peroxisomes; viral replication organelles.

Graphical abstract

Figure 1

SARS-CoV-2 Infection Kinetics in Pulmonary Epithelial Cells (A) Time course of SARS-CoV-2 replication in infected Calu-3 cells (multiplicity of infection [MOI] = 5) as detected by immunofluorescence using a dsRNA antibody (white). Nuclear DNA was stained with DAPI (blue). Scale bar, 40 μm. (B) Percentage of dsRNA⁺ cells quantified from (A). (C and D) Intra- and extracellular viral RNA levels measured by RT-qPCR. (E) Extracellular infectivity measured by plaque assay. Means and SDs of three independent experiments are shown in (B)–(E). (F) Transmission electron microscopy images of 70-nm-thin sections of resin-embedded Calu-3 cells infected with SARS-CoV-2 (MOI = 5) and imaged at the indicated time points after infection. Abbreviations are as follows: DMVs, double-membrane vesicles; C, connectors; LD, lipid droplet; Gg, glycogen granules. Color coding is as follows: orange arrow heads, completed virions; yellow arrowhead, budding virion. Areas in yellow rectangles are magnified in the corresponding panels marked with roman letters. Red dotted lines indicate regions with accumulations of DMVs. (G) Relative frequency distribution of DMV diameters determined at the different time points after infection. Gaussian fits are shown as overlay. n = 43, 40, and 48 DMVs for 6 h, 12 h, and 48 h after infection, respectively.

Figure 2

FIB-SEM Analysis of Whole-Cell Volume of a SARS-CoV-2-Infected Cell Reveals a Network of DMVs and ER Calu-3 cells were infected with SARS-CoV-2 (MOI = 5) for 24 h before being fixed and prepared for FIB-SEM analysis. (A) Two different slices through the cell volume. Note the tight association of the infected cell in the middle with the neighboring cells, giving the infected cell an hourglass-like shape, shown at the top. (B) 3D rendering of the infected cell. The color code of subcellular structures is depicted on the bottom of the figure. (C) Zoom-in of the area indicated with rectangle in (B) showing a cluster of DMVs. (D) Detail of DMV-ER connections (i). DMVs are shown in red, membrane connectors are shown in citrus. (ii) is the same as in (i) but with high-level transparencies for DMVs and ER regions, except the areas in contact with the ER connectors. In (iii and iv) are two orthogonal slices showing the raw data of the same region of the respective left panel. Scale bars, 2 μm in (A) and (B); 500 nm in (C) and (D). See also Figure S1 and Video S1.

Figure 3

High-Resolution Analysis of ER-DMV Inter-Connectivity and Selective Recruitment of ER-Resident Proteins to Sites of Viral Replication Organelles Electron tomography and 3D rendering of SARS-CoV-2-infected Calu-3 cells (MOI = 5) harvested 12 h after infection. (A) Slice through the tomogram. (B) Same region as in (A) with superimposed rendering of cellular and viral organelles. The color code of visualized structures for this and subsequent panels is given in the lower left of the figure. (C) 3D reconstruction of the area indicated with yellow rectangle in (B). (D) Magnified view of DMVs (red) in close contact with membrane connectors (citrus). (E) Magnified view of a DMV in close contact with the ER (green). An ER connector forming a hook is also visible (bottom right). (F) Consecutive slices of a tomogram depicting two adjacent DMVs sharing the outer-membrane with the ER. (G) Side view of the 3D rendering. (H) Orthogonal slices of a tomogram depicting a membrane connector in contact with a DMV and with the ER. A superposition of rendered DMV and ER is shown on the right. (I) 3D rendering view of the DMV and its connectivity to the ER. Scale bars, 200 nm. (J) A549-ACE2 cells were infected with SARS-CoV-2 for 16 h and fixed and stained with primary antibodies of the given specificities. DNA was stained with DAPI (blue). Single slices through deconvoluted z stacks are shown. Scale bar, 10 μm. (K and L) Consecutive slices (K) and 3D rendering (L) of membrane connector bending to form a double-membrane spherule. Red arrows point to double membrane spherules. Scale bars, 200 nm. See also Figure S2 and Videos S2, S3, and S4.

Figure 4

Spatial Coupling of SARS-CoV-2 Replication and Assembly Sites Mediated by Close Proximity of DMVs, Vesicular-Tubular Compartment and Golgi Apparatus (A–F) Electron tomography and 3D rendering of SARS-CoV-2-infected Calu-3 cells (MOI = 0.5) harvested 24 h after infection. (A) Slice through the tomogram. (B) Same region as in (A) with superimposed rendering of cellular and viral organelles that are specified on the bottom of the figure. (C) 3D rendering of organelles visualized in (A). (D–F) Zoom-in view of the vesicular-tubular compartment (VTC) (cyan) and Golgi apparatus (dark blue) with budding virions (yellow), and fully assembled virions (orange). Scale bars, 200 nm. (G) Time course of Golgi fragmentation in SARS-CoV-2-infected (MOI = 5) A549-ACE2 cells. Asterisks indicate infected cells. Scale bar, 10 μm. (H) Quantification of images in (G). For each cell, the largest Golgi fragment was calculated. Each dot indicates the mean values from at least 30 individual cells. Mean and SD of triplicate experiments are shown. p value was calculated with Student’s t test. ^∗∗ = p < 0.01. See also Figures S1, S2, and S4 and Video S5.

Figure 5

A Network of Intermediate Filaments Surrounds SARS-CoV-2 Replication Organelles (A) A549-ACE2 cells were infected with SARS-CoV-2 for 16 h (MOI = 5), fixed and stained with antibodies of the given specificities. DNA was stained with DAPI (blue). A single slice through a deconvolved z stack is shown. The regions in the yellow boxes are magnified in the insets on the left. Scale bar, 10 μm. (B) Cells infected as in (A) were fixed and stained with antibodies directed against dsRNA, the viral replication intermediate, and vimentin. Images were taken by using an Abberior instruments STED microscope. Z stacks comprising whole cells were acquired. Selected slices through the stack are shown in (i). A middle slice with dsRNA (green) and vimentin (gray) signals is shown in (ii). The region in the yellow box is magnified in (iii). A 3D-rendered model of the dsRNA (green) and vimentin (red) signals is shown in (iv). (C) Live cell imaging of SARS-CoV-2-infected A549-ACE2 cells transiently expressing an mCherry-tagged vimentin protein (magenta) and a GFP-NLS-tagged SARS-CoV-2 fluorescent reporter (the structure of this reporter is given on the top). Infected cells show nuclear translocation of the GFP-NLS reporter. Scale bar, 20 μm. Abbreviations are as follows: NLS, nuclear localization sequence; GFP, green fluorescent protein; TM, transmembrane region of Sec61β. The black arrowhead represents the SARS-CoV-2 3C-like protease cleavage site. (D) Frequency distribution of the GFP-NLS nuclear translocation (green bars), vimentin peri-nuclear accumulation (red bars) and infected cell death (gray bars) events. Gaussian fit of each dataset is shown. See also Figure S5 and Videos S6 and S7.

Figure 6

Important Role of the Cytoskeleton for Productive SARS-CoV-2 Replication and Virus Particle Production (A) Compounds that alter the cytoskeletal network were tested on Vero E6 cells infected with SARS-CoV-2. Infection was allowed to proceed for 2 h prior to addition of given concentrations of the indicated compounds. Cells were fixed at 8 h after infection and dsRNA as well as the indicated cytoskeleton elements were detected by immunofluorescence microscopy using specific antibodies. (B) Cell viability after 6 h treatment with the indicated compounds as determined by quantification of intracellular ATP levels. (C) Percentage of infected cells as determined by dsRNA staining of cells treated as in (A). (D) Amounts of infectious SARS-CoV-2 released into the culture supernatant of cells treated as in (A) was quantified by using plaque assay. (C) and (D) show means and SDs; each dot represents the mean of technical triplicates (C) or duplicates (D).