key: cord-0773076-lf7ow8f2 authors: Cortese, Mirko; Lee, Ji-Young; Cerikan, Berati; Neufeldt, Christopher J.; Oorschot, Viola M.J.; Köhrer, Sebastian; Hennies, Julian; Schieber, Nicole L.; Ronchi, Paolo; Mizzon, Giulia; Brey, Inés Romero; Santarella-Mellwig, Rachel; Schorb, Martin; Boermel, Mandy; Mocaer, Karel; Beckwith, Marianne S.; Templin, Rachel M.; Gross, Viktoriia; Pape, Constantin; Tischer, Christian; Frankish, Jamie; Horvat, Natalie K.; Laketa, Vibor; Stanifer, Megan; Boulant, Steeve; Ruggieri, Alessia; Chatel-Chaix, Laurent; Schwab, Yannick; Bartenschlager, Ralf title: Integrative imaging reveals SARS-CoV-2 induced reshaping of subcellular morphologies date: 2020-11-17 journal: Cell Host Microbe DOI: 10.1016/j.chom.2020.11.003 sha: 4534296f970740e8461f19ec6fe74c28f240f5ef doc_id: 773076 cord_uid: lf7ow8f2 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 data-sets of SARS-CoV-2 infected cells for download and smooth online visualization. As of end of September 2020, the ongoing COVID-19 pandemic has caused almost one million 56 fatalities and has affected more than 30 million individuals with confirmed infection worldwide 57 (https://covid19.who.int/). While a second wave of infections is occuring in many countries, the 58 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of this 59 disease, is expected to become seasonal. Thus, there is an urgent need to develop and implement demonstrating that DMVs are the sites of viral RNA synthesis. Consistently, a pore-like opening 104 spanning the two membrane layers of DMVs has been reported very recently, consistent with 105 release of newly synthesized RNA from the DMV interior into the cytoplasm (Wolff et al., 2020) . 106 While these studies show that SARS-CoV-2 infection induces DMV formation as sites of viral 107 RNA replication, the biogenesis of these structures and their link to subcellular compartments is 108 poorly defined. Moreover, although SARS-CoV-2 infection is highly cytopathic, the impact of 109 the virus on integrity and morphology of cellular organelles has not been established. 110 In this study, we employed a combination of light and electron microscopy approaches to obtain Table S1 ). Subsequently, we applied a bottom-up approach, Figure S1 ; Video S1). Using this method, in which a small fraction of one dataset is 161 employed to train a machine learning-based approach for the recognition of defined and known 162 organelles, we segmented the cellular elements from one infected cell and one mock cell, 163 including the nucleus, the mitochondrial network and ER, as well as viral elements, such as the 164 DMVs and DMVs-associated ER membranes (Figure 2B-D; Figure S1 ; Video S1). In addition, (Table S1 ). This large dataset allowed us to identify different In addition to conventional assembly at the VTC and Golgi sites, assembly events were also 271 observed at electron-dense vesicles containing large numbers of virions ( Figure S4F , panels i-iii). Moreover, assembled virions were also present in multivesicular bodies ( Figure S4F , iv). Such CoV-2 replication, we treated infected cells with compounds altering cytoskeleton integrity and 318 dynamics ( Figure 6A ). For this we used drug concentrations that did not induce cytotoxicity as 319 determined by quantification of intracellular ATP levels ( Figure 6B ). We observed a robust The mechanism underlying DMV biogenesis is poorly understood. Close examination of ~900 368 DMVs from more than 200 tomograms identified single openings in just a few of them ( Figure 369 S2D; Table S1 ). While we cannot exclude that membrane integrity was compromised during 370 sample preparation, it is tempting to speculate that DMV openings might represent transition (Figure 4 ; Figure S1 and Figures S4D and S4E) . Figure S1 and Video S1. Table S1 . Sections were analyzed with a JEOL 743 2010 plus transmission electron microscope equipped with a Matataki sCMOS camera Electron tomography (sample acquisition, reconstruction and rendering) After 747 post-staining, screening images were acquired at 1000× and 3000× magnification at points evenly 748 distributed across one section. This was done using advanced SerialEM functionality at a JEM 749 2100 Plus electron microscope (JEOL Ltd equipped with a Gatan OneView US), precise target positions were manually selected within the previously defined regions and 754 acquired by dual axis tomography (-60° to +60° per axis; increment: 1°) at 15,500× magnification 755 (1.55 nm/px). The tilt series were Selected tomograms or parts thereof were manually segmented in Amira-Avizo 758 software Version 2020.1, using the magic wand and the brush segmentation tools Sample preparation for FIB-SEM Calu-3 cells were grown on 35 mm diameter MatTek dishes with glass bottom and were prepared 763 (infection and fixation) as described above for TEM. Extra post-staining steps were performed 764 (the so called OTO post-staining). Cells were post-fixed with osmium-ferricyanide Severe acute respiratory 834 syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles Multicut brings automated neurite segmentation closer to human 838 performance Microscopy Image Browser: A 841 Platform for Segmentation and Analysis of Multidimensional Datasets The Global Phosphorylation Landscape of SARS-845 CoV-2 Infection Comparative tropism, replication kinetics, and cell damage profiling of 849 SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and 850 laboratory studies of COVID-19: an observational study Dense Volumetric Segmentation from Sparse Annotation. Paper presented at: Medical Image Computing 854 and Computer-Assisted Intervention -MICCAI Peroxisome Plasticity at the 857 Virus-Host Interface Ultrastructural Characterization of Zika Virus Replication 861 Factories Peroxisomes are signaling platforms for antiviral innate immunity Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin 869 intermediate filaments A SARS-CoV-2 protein interaction map reveals targets for 873 drug repurposing Membrane 877 rearrangements mediated by coronavirus nonstructural proteins 3 and 4. Virology 458-459 AMST: 883 Alignment to Median Smoothed Template for Focused Ion Beam Scanning Electron Microscopy Image 884 Stacks SARS-CoV-2 Cell Entry Depends on ACE2 and 888 TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Clinical features of patients infected with 2019 novel coronavirus in Wuhan SARS-CoV-2 structure and replication 895 characterized by in situ cryo-electron tomography. bioRxiv SARS-coronavirus replication is supported by a 899 reticulovesicular network of modified endoplasmic reticulum Computer visualization of three-dimensional 902 image data using IMOD Dengue and Zika viruses subvert reticulophagy by NS2B3-905 mediated cleavage of FAM134B Drishti: a volume exploration and presentation tool Distinctive Image Features from Scale-Invariant Keypoints Effect of Systemic Inflammatory Response to SARS-CoV-2 on 914 Lopinavir and Hydroxychloroquine Plasma Concentrations Automated electron microscope tomography using robust prediction of 917 specimen movements Automated tilt series alignment and tomographic 920 reconstruction in IMOD Chapter Eight -The Use of Withaferin A to Study 923 Intermediate Filaments Atlastin Endoplasmic Reticulum-Shaping Proteins 928 Facilitate Zika Virus Replication The cytoskeleton in cell-autonomous immunity: structural 931 determinants of host defence SARS-CoV-2 infection induces a pro-inflammatory 935 cytokine response through cGAS-STING and NF-κB. bioRxiv ER-shaping atlastin proteins act as central hubs to 939 promote flavivirus replication and virion assembly SARS-coronavirus-2 replication in Vero E6 943 cells: replication kinetics, rapid adaptation and cytopathology A Threshold Selection Method from Gray-Level Histograms Antiviral Innate Immune Response Interferes with the Formation of Replication-950 Associated Membrane Structures Induced by a Positive-Strand RNA Virus A versatile reporter system to monitor virus infected cells and its application 954 to dengue virus and SARS-CoV-2. bioRxiv Microscopy-based assay for semi-quantitative detection of SARS-CoV-2 specific 958 antibodies in human sera. bioRxiv Architecture and biogenesis of plus-strand RNA virus replication 961 factories Coronaviridae: The viruses and their replication Role of 966 microtubules in the organization and localization of the Golgi apparatus Fiji: an open-source platform for biological-image 970 analysis NIH Image to ImageJ: 25 years of image 973 analysis Software tools for 976 automated transmission electron microscopy A unifying structural and functional model of the coronavirus 980 replication organelle: Tracking down RNA synthesis The hallmarks of COVID-19 disease Whole-body integration of gene expression and single-987 cell morphology. bioRxiv Role of microtubules in the 990 distribution of the Golgi apparatus: effect of taxol and microinjected anti-alpha-tubulin antibodies A molecular pore spans the double 995 membrane of the coronavirus replication organelle. bioRxiv Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Surface 1001 vimentin is critical for the cell entry of SARS-CoV Highlights • Integrative imaging approaches reveal SARS-CoV-2 induced cellular alterations • SARS-CoV-2 extensively remodels the cellular endomembrane system • Pharmacological inhibition of cytoskeleton remodeling restricts viral replication • We provide a comprehensive repository of virus-induced ultrastructural cell changes by SARS-CoV-2 in lung cells. Accumulation of ER-derived double membrane vesicles, the viral replication organelle, occurs concomitantly with cytoskeleton remodeling and Golgi fragmentation. Pharmacological alteration of cytoskeleton dynamics restricts viral replication and spread