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

•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 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. 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. As of end of September 2020, the ongoing COVID-19 pandemic has caused almost one million fatalities and has affected more than 30 million individuals with confirmed infection worldwide (https://covid19.who.int/). A second wave of infections is occurring in many countries and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of this disease, is expected to become seasonal. Thus, there is an urgent need to develop and implement both prophylactic and therapeutic strategies against this virus. Tremendous efforts are being deployed to rapidly develop a safe-guarding vaccine with no less than 150 candidates currently under evaluation. Therefore, much attention has been drawn to the study of virion structure and potential surface epitopes relevant for induction of neutralizing antibodies (Chu et al., 2020Chu H. Chan J.F.-W. Yuen T.T.-T. Shuai H. Yuan S. Wang Y. Hu B. Yip C.C.-Y. Tsang J.O.-L. Huang X. et al.Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study.Lancet Microbe. 2020; 1: e14-e23Abstract Full Text Full Text PDF PubMed Google Scholar; Wrapp et al., 2020Wrapp D. Wang N. Corbett K.S. Goldsmith J.A. Hsieh C.L. Abiona O. Graham B.S. McLellan J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science. 2020; 367: 1260-1263Crossref PubMed Scopus (82) Google Scholar). In stark contrast, much less is known about how SARS-CoV-2 drives pathogenesis, but it is becoming clear that disease severity is determined by two parameters. These are a predominantly inflammatory cytokine response triggered by the virus and direct cytopathogenicity of SARS-CoV-2, leading to death of infected cells by a so far poorly characterized mechanism (Chu et al., 2020Chu H. Chan J.F.-W. Yuen T.T.-T. Shuai H. Yuan S. Wang Y. Hu B. Yip C.C.-Y. Tsang J.O.-L. Huang X. et al.Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study.Lancet Microbe. 2020; 1: e14-e23Abstract Full Text Full Text PDF PubMed Google Scholar; Huang et al., 2020Huang C. Wang Y. Li X. Ren L. Zhao J. Hu Y. Zhang L. Fan G. Xu J. Gu X. et al.Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.Lancet. 2020; 395: 497-506Abstract Full Text Full Text PDF PubMed Scopus (29361) Google Scholar; Tang et al., 2020Tang D. Comish P. Kang R. The hallmarks of COVID-19 disease.PLoS Pathog. 2020; 16: e1008536Crossref PubMed Scopus (256) Google Scholar). Knowledge about viral cytopathogenicity requires detailed insights into how SARS-CoV-2 replicates in and alters its host cell. Information gained in this area is expected to foster the development of innovative therapy mitigating disease severity. However, thus far such insights have been mostly inferred from studies on related betacoronaviruses such as the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-1, the mouse hepatitis virus (MHV) model system, or some endemic coronaviruses causing common cold. Although some approved drugs are considered for repurposing strategies (e.g., hydroxychloroquine or combination therapy of lopinavir-ritonavir) because of their antiviral activity in cell culture, the clinical benefit of these drugs in terms of survival outcome and severity of symptoms is disputed (Marzolini et al., 2020Marzolini C. Stader F. Stoeckle M. Franzeck F. Egli A. Bassetti S. Hollinger A. Osthoff M. Weisser M. Gebhard C.E. et al.Effect of Systemic Inflammatory Response to SARS-CoV-2 on Lopinavir and Hydroxychloroquine Plasma Concentrations.Antimicrob. Agents Chemother. 2020; 64 (e01177-20)Crossref PubMed Scopus (41) Google Scholar). Therefore, there is a need to better understand the biological mechanisms driving the SARS-CoV-2 replication cycle in order to identify therapeutic targets and develop highly efficient drugs suppressing viral replication and virus-induced cell death. After viral entry initiated by the binding of Spike (S) viral protein to cell surface receptors, the best-studied one being angiotensin I converting enzyme 2 (ACE2), and S processing by cellular proteases (Hoffmann et al., 2020Hoffmann M. Kleine-Weber H. Schroeder S. Kruger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. et al.SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.Cell. 2020; 181: 271-280Abstract Full Text Full Text PDF PubMed Scopus (11809) Google Scholar), the 30 kb-long positive-strand RNA genome is released into the cytoplasm. There, the two large open reading frames, 1a and, via ribosomal frameshifting, 1a/b are translated giving rise to the replicase (rep) polyproteins rep1a and rep1a/1b, respectively. These viral products are cleaved by viral proteases to generate 16 mature nonstructural proteins (nsps), most of them constituting the replication-transcription complex responsible for viral RNA synthesis. During replication, several sub-genomic RNA species are generated encoding for four structural proteins and multiple accessory proteins. The structural proteins membrane (M), nucleocapsid (N), envelope (E), and S, together with the genomic RNA, drive the assembly of new virus particles, which in the case of other coronaviruses bud into the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). Virions are transported through the secretory pathway and are released out of the cell (Perlman and Masters, 2020Perlman S. Masters P.S. 7th Edition. Coronaviridae: The viruses and their replication. Vol. 1. 2020Google Scholar). Like all positive-strand RNA viruses (Paul and Bartenschlager, 2013Paul D. Bartenschlager R. Architecture and biogenesis of plus-strand RNA virus replication factories.World J. Virol. 2013; 2: 32-48Crossref PubMed Google Scholar), SARS-CoV-2 induces a remodeling of cellular endomembranes to form viral replication organelles (vROs). These structures are thought to create a microenvironment conducive to RNA synthesis by allowing the enrichment of metabolites, viral enzymes and cofactors, and by protecting viral RNA from degradation and sensing by pattern recognition receptors of the innate immune system. Coronavirus replication organelles (ROs) are composed predominantly of double-membrane vesicles (DMVs) that are most likely derived from the endoplasmic reticulum (ER) (Klein et al., 2020Klein S. Cortese M. Winter S.L. Wachsmuth-Melm M. Neufeldt C.J. Cerikan B. Stanifer M.L. Boulant S. Bartenschlager R. Chlanda P. SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography.bioRxiv. 2020; https://doi.org/10.1101/2020.06.23.167064Crossref Scopus (0) Google Scholar; Knoops et al., 2008Knoops K. Kikkert M. Worm S.H. Zevenhoven-Dobbe J.C. van der Meer Y. Koster A.J. Mommaas A.M. Snijder E.J. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.PLoS Biol. 2008; 6: e226Crossref PubMed Scopus (704) Google Scholar; Snijder et al., 2020Snijder E.J. Limpens R.W.A.L. de Wilde A.H. de Jong A.W.M. Zevenhoven-Dobbe J.C. Maier H.J. Faas F.F.G.A. Koster A.J. Bárcena M. A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis.PLoS Biol. 2020; 18: e3000715Crossref PubMed Scopus (237) Google Scholar). The interior of these structures contains double-stranded (ds)RNA, the presumed viral replication intermediate, and de novo synthesized RNA, demonstrating that DMVs are the sites of viral RNA synthesis. A pore-like opening spanning the two membrane layers of DMVs has been reported very recently, consistent with release of newly synthesized RNA from the DMV interior into the cytoplasm (Wolff et al., 2020Wolff G. Limpens R.W.A.L. Zevenhoven-Dobbe J.C. Laugks U. Zheng S. de Jong A.W.M. Koning R.I. Agard D.A. Grünewald K. Koster A.J. et al.A molecular pore spans the double membrane of the coronavirus replication organelle.bioRxiv. 2020; https://doi.org/10.1101/2020.06.25.171686Crossref Google Scholar). Although these studies show that SARS-CoV-2 infection induces DMV formation as sites of viral RNA replication, the biogenesis of these structures and their link to subcellular compartments is poorly defined. Moreover, although SARS-CoV-2 infection is highly cytopathic, the effect of the virus on integrity and morphology of cellular organelles has not been established. In this study, we employed a combination of light and electron microscopy approaches to obtain an integrative view of the 3D architecture of SARS-CoV-2-induced vROs, their inter-relation with subcellular compartments, and the effect of viral infection on cellular organelles. We show whole-cell 3D reconstructions demonstrating profound morphological remodeling of multiple membranous organelles such as fragmentation of the Golgi and recruitment of peroxisomes to vROs. In addition, using live cell imaging in combination with a sensor monitoring productive infection and replication, we show that DMV clusters are delimited by a reorganized “cage-like” vimentin network and that pharmacological inhibition of vimentin blocks viral replication. In cellulo electron tomography and focused ion beam scanning electron microscopy (FIB-SEM) data unveiled a network of interconnected DMVs that are tethered to the endoplasmic reticulum (ER) by membrane connectors, providing insights into DMV biogenesis and their role in coordinating the different steps of SARS-CoV-2 replication. Altogether, our study provides a comprehensive 3D view of the SARS-CoV-2 replication cycle and alterations of cellular organelles most likely contributing to cytopathogenicity of the virus and possibly serving as target for urgently needed therapeutic strategies. Human pulmonary epithelial Calu-3 cells are known to be permissive to SARS-CoV-2 and therefore were used as model system to study the morphological remodeling of the cell induced by viral infection. From 6 h after infection onward, SARS-CoV-2+ cells as well as intra- and extracellular viral RNA and infectious virus released into the cell culture supernatant became detectable (Figures 1A–1E ). Thus, a full replication cycle can be completed within less than 6 h in Calu-3 cells. At 12 and 24 h after infection, the number of infected cells increased up to 70% (Figure 1B), concomitant with an increase of intra- and extracellular viral RNA as well as virus titers as determined by infectivity assay (Figures 1C–1E). Ultrastructural analysis performed by transmission electron microscopy (TEM) revealed a parallel appearance of DMVs, becoming detectable as sporadic clusters of small-sized DMVs (diameter 185 nm ± 28 nm) at 6 h after infection and increasing in abundance and diameter (298 nm ± 42 nm) until 24 h after infection (Figures 1F and 1G). In some cases, large areas covered with glycogen granules, in close proximity of lipid droplets, and membranous cisternae were present within infected cells (Figure 1Fii, bottom and 1Fiii, middle). Virions assembling within the Golgi compartments as well as few extracellular virions were observed, starting at 6 h after infection and also increasing in abundance at later time points (Figure 1Fii top and 1Fiv, right). Notably, ER tubules with collapsed luminal space were frequently observed in close proximity to DMVs (Figure 1Fiii, right). Such structures are reminiscent of the so-called “zippered ER” or “convoluted membranes” observed in cells infected with other coronaviruses (Snijder et al., 2020Snijder E.J. Limpens R.W.A.L. de Wilde A.H. de Jong A.W.M. Zevenhoven-Dobbe J.C. Maier H.J. Faas F.F.G.A. Koster A.J. Bárcena M. A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis.PLoS Biol. 2020; 18: e3000715Crossref PubMed Scopus (237) Google Scholar). In some instances, these structures appear to connect the rough ER to the DMVs (Figure 1Fi, bottom left and 1Fiii, right). To obtain a global view of the cellular alterations induced by SARS-CoV-2 infection, we applied FIB-SEM analysis on infected Calu-3 cells. The full volumes of three infected cells (infection being determined by the presence of DMVs), and two mock-inoculated cells that served as reference, were acquired (the complete datasets are made available for download on EMPIAR—ID 10490, and are viewable in an interactive manner via MoBIE—see STAR Methods and Table S1). Subsequently, we applied a bottom-up approach, efficiently combining semi-automated and automated segmentation based on the multicut pipeline (Beier et al., 2017Beier T. Pape C. Rahaman N. Prange T. Berg S. Bock D.D. Cardona A. Knott G.W. Plaza S.M. Scheffer L.K. et al.Multicut brings automated neurite segmentation closer to human performance.Nat. Methods. 2017; 14: 101-102Crossref PubMed Scopus (90) Google Scholar) (see STAR Methods) to achieve deep-segmentation of the dataset (Figures 2 and S1; Video S1). Using this method, in which a small fraction of one dataset is employed to train a machine-learning-based approach for the recognition of defined and known organelles, we segmented the cellular elements from one infected cell and one mock cell, including the nucleus, the mitochondrial network, and ER, as well as viral elements, such as the DMVs and DMV-associated ER membranes (Figures 2B–2D and S1; Video S1). In addition, manual segmentation was applied for the Golgi apparatus in the infected cell (Figures 2B and 2C) and some instances in the mock cell where the upscaling was performed by using deep learning (Figure S1). Rendering and 3D visualization of the whole segmented dataset revealed an intricate network of DMVs embedded in an ER matrix. The majority of DMVs accumulated on the side of the cell attached to the culture substrate (Figure 2B), although groups of DMVs were present throughout the cell and always in contact with the ER network. Closer inspection showed that the ER tubules with narrow luminal space linked several DMVs together, also connecting them to the larger ER network (Figures 2C and 2D; Video S1). Therefore, we have named these structures “ER connectors.” eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyMzA2MjdkMjJmYTYxMjU4MGMzZGE5MjVlMGYyN2FiZSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjkwMzQ5NDAxfQ.B9JoVgFzfnJA6vHYK28comP4vO2htyI408XOc2EUhsSJkebX0WaWdhD5mKNozd_Cx8f-htAP-nRBkf5JPrdaevK96n1sLrndctUD7XnC96Uzzsd4nRcV4LMZHcFjIvYznQwNWS3bWP6iDYnb1oVVhkQrEkPD7uKBs-bW7Fodzpy2HuM_p_huQEdxliRnwR1O4oPeuD0gJtR86AM7hw2lcYmZH-ZE2uar2sfbsdRFUDyc9Lum7XzJFk1jt8HWk2b6dhJFQhfQ0nUpSjyVWsz4pmuNckDJ9c5gxYx_U2ZDq6uT6jL-TqmWoLF3NekzWIBnZdHd8ztmZCMKA9NIpbjmIg Download .mp4 (17.6 MB) Help with .mp4 files Video S1. FIB-SEM Analysis of a SARS-CoV-2-Infected Cell, Related to Figure 2 To gain insight into the biogenesis of the DMVs, we determined their 3D architecture by high-resolution electron tomography analysis of SARS-CoV-2-infected Calu-3 cells. We processed a total of 13 tomograms from uninfected cells and 7, 85, and 153 dual-tilt tomograms of cells fixed at 6, 12, and 24 h after infection, respectively (Table S1; the complete dataset is made available for download on EMPIAR—ID 10490 and can be visualized through the MoBIE Fiji plugin; see Figure S2A and STAR Methods). For each tomogram, we identified and classified the cellular and viral structures present (Table S1). This large dataset allowed us to identify different topological compositions of the SARS-CoV-2 RO. Among all virus-induced structures, DMVs were the most abundant. The average diameter, calculated at the DMVs’ equator, was 291 ± 48 nm for the 24 h time point, in agreement with the results from TEM (Figures S2B and 1G, respectively). Smooth ER connectors were often found in close apposition to the DMVs’ outer membranes (Figures 3A–3E ). In some cases, DMVs were embedded into the rough ER such as the DMV outer membrane was contiguous to the ER membrane, and several DMVs were associated to the same ER branch (Figures 3F and 3G, respectively; Video S2). This interconnection, together with the presence of ribosomes on the DMVs’ outer membrane (Figure S2C) suggests that DMVs originate from the rough ER. Additionally, smooth ER connectors were seen linking DMVs to the rough ER (Figures 3A–3D, 3H, and 3I; Video S3), corroborating the observations made by the FIB-SEM analysis. With very low frequency, we observed an opening in the DMV membranes (Figure S2D) that connected the DMV interior to the cytosol. Although the presence of such openings could allow for the exchange of metabolites and for the release of newly synthesized viral RNAs into the cytoplasm, their low frequency argues for a very short-lived transition state prior to complete closure of the DMV membrane. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJhZjljZmRkY2M3ZThkZDc0MDkwMzc2ZjJhNTU3OWVhNiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjkwMzQ5NDAxfQ.UoYkrsQgK9i0awQces_XjBTpkpLlq1T1WEyoyhGhRHFTO6eJLLClSHdKJEYUg_EI3P9dSgxeevdCrcoeifq5tVuyIfq2M-gCQHBvDySCqT-3qdgah1sOS3PcSJbOw4Kdumi-TYw0MsFGO0u_s79g6ur5-j8yt-qIPa5HhS5mjD_1Da_CXUu3ATqHfSbIz9GEwy2dgLsFwhkUpegdHMrboB3aDFPYjXC6QODu6DPLSFZdKLu2MiNdH9S54MFBO6EdXP9zN-VfU3HlsuTWamiRCRr1PeNmBprZYOh5gBOg7U3zCl1XTW1dJGnt5MUjLP1eBcrNhmYu7vrTXn0U7MFOvQ Download .mp4 (7.2 MB) Help with .mp4 files Video S2. Electron Tomogram of DMVs and Their Connection with the ER, Related to Figure 3 eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJiMTFhYWJkYjUyMGE2MzdhMTNkYjFjMGM0YjRmYmY3MiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjkwMzQ5NDAxfQ.OHuRs54xjpBwgu8pjCXOkeRE2dPTYOYN99YldYEx1XXnk_c5KEyKzoseKy8w_4A797lcDf5Vtx5HU7lvWcPhXIV6k1rYgJruQEAqAoDJxlKUcL_ZllZgJ6pEHQG1vr_seNsEgnyCsCIO5g7HavoUnDJw3F0m3tLEvZ9ZHZuK0XLhSmrwccrmP-BhIyQwSoAyk53PXFwKDse5Khv_cNEjDan6Uu4WRoMikcS3n1qYXVr-_HxhkDXkRdDWneyzNj3ExhrDNWIe-tkK_HhqccIFoqUUQOyN6UwkDvmnWBy2q1FC0r9CVPTr9-twISA89Jk0IF8WUs38ShwqI0vlahM7Xg Download .mp4 (8.38 MB) Help with .mp4 files Video S3. Electron Tomogram of DMVs in Contact with ER Connectors, Related to Figure 3 Consistent with recent reports (Klein et al., 2020Klein S. Cortese M. Winter S.L. Wachsmuth-Melm M. Neufeldt C.J. Cerikan B. Stanifer M.L. Boulant S. Bartenschlager R. Chlanda P. SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography.bioRxiv. 2020; https://doi.org/10.1101/2020.06.23.167064Crossref Scopus (0) Google Scholar; Ogando et al., 2020Ogando N.S. Dalebout T.J. Zevenhoven-Dobbe J.C. Limpens R.W.A.L. van der Meer Y. Caly L. Druce J. de Vries J.J.C. Kikkert M. Bárcena M. et al.SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology.J. Gen. Virol. 2020; 101: 925-940Crossref PubMed Scopus (279) Google Scholar), DMV-DMV contacts were also observed, either through funnel-like junctions between two DMVs (Figure S2E) or by the formation of tabs and indentations in adjacent DMVs that resemble jigsaw puzzle pieces (Figure S2F). Ultimately, these membrane bending events might generate fused DMVs (Figure S2G) consisting of multiple vesicles sharing the same outer membrane, or multimembrane vesicles formed by DMVs that have engulfed either a single- or a double-membrane vesicle (Figure S2H). Re-organization of the ER network was also confirmed by confocal microscopy of SARS-CoV-2-infected A549 lung epithelial cells overexpressing ACE2. We observed localization of the tubular ER protein Reticulon 3 (RTN3) in the perinuclear region containing high amounts of double-stranded RNA, a marker of viral replication, and the viral protein nsp3 (Figure 3J). Conversely, the Sec61β subunit of the translocon was mostly excluded from this region, indicating that despite the close link of DMVs to the ER, the synthesis of transmembrane or secreted proteins is relocated to the vRO periphery (Figure 3J). In our datasets, double-membrane spherules, similar to the ones described for other coronaviruses (Snijder et al., 2020Snijder E.J. Limpens R.W.A.L. de Wilde A.H. de Jong A.W.M. Zevenhoven-Dobbe J.C. Maier H.J. Faas F.F.G.A. Koster A.J. Bárcena M. A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis.PLoS Biol. 2020; 18: e3000715Crossref PubMed Scopus (237) Google Scholar), were only rarely seen in SARS-CoV-2-infected cells. These structures had a diameter of ∼75.5 ± 5.9 nm, an electron-dense interior and were linked to the connectors from which they originated (Figures 3K and 3L; Video S4). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIxZTg0YWRkYjRlMTMzNzZjZDA4YWUxYjcxODI0MDlhMiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjkwMzQ5NDAxfQ.Skmrn8GbzquCQEihIeL3oxea-rHZaHZGoRUK-yHG0aolPIb8MgrWphTjyXHTlNRW2G9yHenWitjuithAKR3OaOKtfADqznSB1YaSN_n4ymr1mMhOPmrYyIc1J2cXOv9Ka96EQ4EP5MAXC2b9kJuWyepQ6KVlNHuoj8jT6S8b0LMxVm6bDHEqZHSI8ELr_6VQtE0OwTsqOCex8dPRL0O9FEzEf_fHSHIBw3oV8F0abYrcWx6wHVFn-1c-3OIn92u_vjxgxtZ51Qm-nQJjaQuTja58x66UDTeB3D9MAaBx_t2qCBTgFpHpt9JK0vweEWeao9N2ZLwcWH0vWmnz3O8-VQ Download .mp4 (7.64 MB) Help with .mp4 files Video S4. Electron Tomogram of Double-Membrane Spherules and their Association with the ER, Related to Figure 3 Altogether, our 3D reconstruction data provide strong evidence that SARS-CoV-2 reorganizes the ER network and alters the morphology of this organelle to generate vROs, which consist predominantly of DMVs, but also include other ER-derived structures such as ER connectors, double-membrane spherules, and multi-membrane vesicles. In addition to the formation of de novo ER-derived organelles, SARS-CoV-2 replication alters the morphology, the number, and the function of several other cellular compartments. Of note, in regions containing DMV clusters, we observed accumulation of peroxisomes in close proximity to the DMVs’ outer membrane (Figures 4A–4C , S3A, andS3B; Video S5). These results were confirmed by confocal microscopy and western blot analyses, showing a redistribution of peroxisomes to double-stranded RNA (dsRNA)+ regions (Figures S3C and S3D) and an increase in the peroxisome-associated protein PMP70 in infected cells (Figure S3E). Consistently, super-resolution microscopy revealed an enclosing of the dsRNA signal by the peroxisomal signal (Figure S3F), supporting the topology observed by electron tomography (Figures 4A–4C). The spatial proximity between peroxisomes and the sites of viral RNA replication (Figures S3A and S3B) argues for a role of peroxisomes in the SARS-CoV-2 replication cycle such as the prevention of oxidative damage to viral RNA or an involvement in lipid metabolism (Cook et al., 2019Cook K.C. Moreno J.A. Jean Beltran P.M. Cristea I.M. Peroxisome Plasticity at the Virus-Host Interface.Trends Microbiol. 2019; 27: 906-914Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) or serving as signaling platform mounting a cytokine response (Dixit et al., 2010Dixit E. Boulant S. Zhang Y. Lee A.S. Odendall C. Shum B. Hacohen N. Chen Z.J. Whelan S.P. Fransen M. et al.Peroxisomes are signaling platforms for antiviral innate immunity.Cell. 2010; 141: 668-681Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI2Yzk0YmM0NjAxMjBhNWE0NzE1NTNhNzNlMWU5NjI3MCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjkwMzQ5NDAxfQ.Ssf9xQaX9Ei6Zjcr1gUuDDiZRdLczPY5E5nB4NKplEOlTDU3Rahlu0VnwOJda4SJkEDJt5hydwIeZYbSnSf4Y1qcfVMDxTJsosfukTOepVwc4cTWhIL9r8_acELkk8z3yYRm-Quie-TKqIabOX2xCmf8nggK0cwr6sMaXmF6cif1EPQ2CN5qLtVogklyLl0kw_FEB3DvZCmCwx-8CMHJ0Sf9Fd69oaqdbjVeYgOv-lmWDseX_XUBzyncdMhMYPOpPQiZCT793ka2bvzXUuv8GQq4_5LIJb7is8u-JJ2gYK-KHisVkiJldp1cqPpSAdbcJCTXa27t53JLCCVSBAZokg Download .mp4 (44.41 MB) Help with .mp4 files Video S5. Electron Tomogram of SARS-CoV-2 Replication Organelle and Spatial Link to Vesicular-Tubular Compartment/Golgi Apparatus Located Assembly Sites, Related to Figure 4 Mitochondria were also altered in SARS-CoV-2-infected cells in several ways. First, they were displaced and accumulated at the periphery of dsRNA+ regions (Figure S3G). Second, mitochondria morphology was altered showing an increase of both intracristal space and matrix density, conferring an electron-dense appearance in transmission electron microscopy (Figure S3H). Third, in infected cells, mitochondria were significantly thinner than in uninfected cells (Figure S3I). Forth, in infected cells, we observed a strong decrease in the total amounts of the mitochondrial ATP synthase subunit 5B (ATP5B), a key factor for cellular energy production (Figures S3J and S3K). Altogether, these results reveal strong perturbation of mitochondria morphology and function, most likely reflecting SARS-CoV-2-induced attenuation of cellular energy metabolism. The high frequency of budding events observed in the Golgi apparatus and the surrounding vesicular membrane compartment indicates that these organelles provide membranes for SARS-CoV-2 assembly (Figures 4D–4F; Video S5). Within these cellular compartments, strings of viral nucleoprotein, corresponding to dark-stained granules were found on bent membranes, which, given the similarities in morphology to fully assembled virions, probably correspond to the early stages of virion budding (Figure S4A). Consistent with previous reports, we also observed fully assembled virions with an average diameter of 80 ± 9.5 nm (Figure S4B) (Klein et al., 2020Klein S. Cortese M. Winter S.L. Wachsmuth-Melm M. Neufeldt C.J. Cerikan B. Stanifer M.L. Boulant S. Bartenschlager R. Chlanda P. SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography.bioRxiv. 2020; https://doi.org/10.1101/2020.06.23.167064Crossref Scopus (0) Google Scholar). The global overview obtained from the large set of tomograms and the FIB-SEM data reveal that assembly sites, corresponding to the Golgi and surrounding vesicles, and DMVs, the RNA replication sites, are in close proximity, suggesting spatiotemporal coordination of the different steps of the SARS-CoV-2 replication cycle (Figures 4A–4F and S1; Videos S1 and S5). In uninfected cells, the secretory compartment showed well-defined morphology. The polarization of the Golgi stacks allowed for reliable identification of the ER-to-Golgi intermediate compartment (ERGIC) area and the presence of clathrin-