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1.
Biochem Biophys Res Commun ; 586: 137-142, 2022 01 01.
Article in English | MEDLINE | ID: covidwho-1520712

ABSTRACT

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.


Subject(s)
COVID-19/metabolism , COVID-19/virology , Host Microbial Interactions/physiology , Membrane Glycoproteins/metabolism , Nuclear Pore Complex Proteins/metabolism , RNA-Binding Proteins/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Active Transport, Cell Nucleus , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Gene Knockdown Techniques , HeLa Cells , Humans , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/genetics , Models, Biological , Nuclear Envelope/metabolism , Nuclear Envelope/virology , Nuclear Pore Complex Proteins/antagonists & inhibitors , Nuclear Pore Complex Proteins/genetics , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factor RelA/metabolism , Viral Nonstructural Proteins/genetics
2.
J Cell Biochem ; 123(2): 155-160, 2022 02.
Article in English | MEDLINE | ID: covidwho-1473858

ABSTRACT

Drug repurposing is an attractive option for identifying new treatment strategies, in particular in extraordinary situations of urgent need such as the current coronavirus disease 2019 (Covid-19) pandemic. Recently, the World Health Organization announced testing of three drugs as potential Covid-19 therapeutics that are known for their dampening effect on the immune system. Thus, the underlying concept of selecting these drugs is to temper the potentially life-threatening overshooting of the immune system reacting to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. This viewpoint discusses the possibility that the impact of these and other drugs on autophagy contributes to their therapeutic effect by hampering the SARS-CoV-2 life cycle.


Subject(s)
Antiviral Agents/pharmacology , Artesunate/pharmacology , Autophagy/drug effects , COVID-19/drug therapy , Drug Repositioning , Imatinib Mesylate/pharmacology , Infliximab/pharmacology , Pandemics , SARS-CoV-2/drug effects , Antidepressive Agents/pharmacology , Antiviral Agents/therapeutic use , Artesunate/therapeutic use , Chloroquine/pharmacology , Drug Development , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/physiology , Endoplasmic Reticulum/virology , Endosomes/drug effects , Endosomes/virology , Humans , Hydroxychloroquine/pharmacology , Imatinib Mesylate/therapeutic use , Infliximab/therapeutic use , Intracellular Membranes/drug effects , Intracellular Membranes/physiology , Intracellular Membranes/virology , Ivermectin/pharmacology , Macrolides/pharmacology , Middle East Respiratory Syndrome Coronavirus/drug effects , Niclosamide/pharmacology , Niclosamide/therapeutic use , RNA, Viral/metabolism , SARS-CoV-2/physiology , Virus Replication
3.
Cells ; 10(10)2021 09 24.
Article in English | MEDLINE | ID: covidwho-1438526

ABSTRACT

Eukaryotic cells contain dynamic membrane-bound organelles that are constantly remodeled in response to physiological and environmental cues. Key organelles are the endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which are interconnected by vesicular traffic through the secretory transport route. Numerous viruses, especially enveloped viruses, use and modify compartments of the secretory pathway to promote their replication, assembly and cell egression by hijacking the host cell machinery. In some cases, the subversion mechanism has been uncovered. In this review, we summarize our current understanding of how the secretory pathway is subverted and exploited by viruses belonging to Picornaviridae, Coronaviridae, Flaviviridae, Poxviridae, Parvoviridae and Herpesviridae families.


Subject(s)
Endoplasmic Reticulum/virology , Golgi Apparatus/virology , Secretory Pathway/physiology , Viruses/isolation & purification , Biological Transport/physiology , Cell Membrane/metabolism , Cell Membrane/virology , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Humans
4.
Viruses ; 13(9)2021 09 09.
Article in English | MEDLINE | ID: covidwho-1411084

ABSTRACT

A variety of immunolabeling procedures for both light and electron microscopy were used to examine the cellular origins of the host membranes supporting the SARS-CoV-2 replication complex. The endoplasmic reticulum has long been implicated as a source of membrane for the coronavirus replication organelle. Using dsRNA as a marker for sites of viral RNA synthesis, we provide additional evidence supporting ER as a prominent source of membrane. In addition, we observed a rapid fragmentation of the Golgi apparatus which is visible by 6 h and complete by 12 h post-infection. Golgi derived lipid appears to be incorporated into the replication organelle although protein markers are dispersed throughout the infected cell. The mechanism of Golgi disruption is undefined, but chemical disruption of the Golgi apparatus by brefeldin A is inhibitory to viral replication. A search for an individual SARS-CoV-2 protein responsible for this activity identified at least five viral proteins, M, S, E, Orf6, and nsp3, that induced Golgi fragmentation when expressed in eukaryotic cells. Each of these proteins, as well as nsp4, also caused visible changes to ER structure as shown by correlative light and electron microscopy (CLEM). Collectively, these results imply that specific disruption of the Golgi apparatus is a critical component of coronavirus replication.


Subject(s)
Endoplasmic Reticulum/virology , Golgi Apparatus/virology , SARS-CoV-2/physiology , Virus Replication , Animals , Chlorocebus aethiops , Coronavirus M Proteins/physiology , Coronavirus M Proteins/ultrastructure , Endoplasmic Reticulum/ultrastructure , Golgi Apparatus/ultrastructure , Humans , Intracellular Membranes/ultrastructure , Intracellular Membranes/virology , Microscopy, Electron , SARS-CoV-2/ultrastructure , Vero Cells , Viral Structural Proteins/physiology , Viral Structural Proteins/ultrastructure
5.
Cell Host Microbe ; 28(6): 853-866.e5, 2020 12 09.
Article in English | MEDLINE | ID: covidwho-1385263

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.


Subject(s)
COVID-19/genetics , Endoplasmic Reticulum/ultrastructure , SARS-CoV-2/ultrastructure , Viral Replication Compartments/ultrastructure , COVID-19/diagnostic imaging , COVID-19/pathology , COVID-19/virology , Cell Death/genetics , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/virology , Humans , Microscopy, Electron , Pandemics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Viral Replication Compartments/metabolism , Virus Replication/genetics
6.
Basic Res Cardiol ; 116(1): 42, 2021 07 05.
Article in English | MEDLINE | ID: covidwho-1293364

ABSTRACT

Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant.


Subject(s)
Endoplasmic Reticulum/virology , Endothelial Cells/virology , SARS-CoV-2/pathogenicity , Angiotensin-Converting Enzyme 2/metabolism , Calnexin/metabolism , Cells, Cultured , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Endothelial Cells/metabolism , Host-Pathogen Interactions , Humans , Membrane Proteins/metabolism , Receptors, Virus/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
7.
J Biol Chem ; 296: 100759, 2021.
Article in English | MEDLINE | ID: covidwho-1219049

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors might also play important roles in SARS-CoV-2 infection, providing new directions for antiviral treatments. GRP78 is a stress-inducible chaperone important for entry and infectivity for many viruses. Recent molecular docking analyses revealed putative interaction between GRP78 and the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (SARS-2-S). Here we report that GRP78 can form a complex with SARS-2-S and ACE2 on the surface and at the perinuclear region typical of the endoplasmic reticulum in VeroE6-ACE2 cells and that the substrate-binding domain of GRP78 is critical for this interaction. In vitro binding studies further confirmed that GRP78 can directly bind to the RBD of SARS-2-S and ACE2. To investigate the role of GRP78 in this complex, we knocked down GRP78 in VeroE6-ACE2 cells. Loss of GRP78 markedly reduced cell surface ACE2 expression and led to activation of markers of the unfolded protein response. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159) selected for its safe clinical profile in preclinical models depleted cell surface GRP78 and reduced cell surface ACE2 expression, as well as SARS-2-S-driven viral entry and SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78 in combination therapy.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Heat-Shock Proteins/genetics , Host-Pathogen Interactions/genetics , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization/drug effects , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Binding Sites , Chlorocebus aethiops , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Gene Expression Regulation , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/metabolism , Humans , Mutation , Protein Binding , Protein Domains , Protein Multimerization , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Signal Transduction , Spike Glycoprotein, Coronavirus/metabolism , Unfolded Protein Response , Vero Cells
8.
Mol Cell ; 81(12): 2656-2668.e8, 2021 06 17.
Article in English | MEDLINE | ID: covidwho-1179919

ABSTRACT

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.


Subject(s)
Antigens, CD/genetics , Host-Pathogen Interactions/genetics , Interferon Regulatory Factors/genetics , Interferon Type I/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics , Animals , Antigens, CD/chemistry , Antigens, CD/immunology , Binding Sites , Cell Line, Tumor , Chlorocebus aethiops , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/virology , GPI-Linked Proteins/chemistry , GPI-Linked Proteins/genetics , GPI-Linked Proteins/immunology , Gene Expression Regulation , Golgi Apparatus/genetics , Golgi Apparatus/immunology , Golgi Apparatus/virology , HEK293 Cells , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Interferon Regulatory Factors/classification , Interferon Regulatory Factors/immunology , Interferon Type I/immunology , Molecular Docking Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/immunology , Signal Transduction , Vero Cells , Viral Proteins/chemistry , Viral Proteins/immunology , Virus Internalization , Virus Release/genetics , Virus Release/immunology , Virus Replication/genetics , Virus Replication/immunology
9.
Cell Microbiol ; 23(8): e13328, 2021 08.
Article in English | MEDLINE | ID: covidwho-1142875

ABSTRACT

Annulate lamellae (AL) have been observed many times over the years on electron micrographs of rapidly dividing cells, but little is known about these unusual organelles consisting of stacked sheets of endoplasmic reticulum-derived membranes with nuclear pore complexes (NPCs). Evidence is growing for a role of AL in viral infection. AL have been observed early in the life cycles of the hepatitis C virus (HCV) and, more recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suggesting a specific induction of mechanisms potentially useful to these pathogens. Like other positive-strand RNA viruses, these viruses induce host cells membranes rearrangements. The NPCs of AL could potentially mediate exchanges between these partially sealed compartments and the cytoplasm. AL may also be involved in regulating Ca2+ homeostasis or cell cycle control. They were recently observed in cells infected with Theileria annulata, an intracellular protozoan parasite inducing cell proliferation. Further studies are required to clarify their role in intracellular pathogen/host-cell interactions.


Subject(s)
Host-Pathogen Interactions/physiology , Organelles/microbiology , Organelles/parasitology , Animals , COVID-19 , Cytoplasm/virology , Endoplasmic Reticulum/microbiology , Endoplasmic Reticulum/parasitology , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum/virology , Humans , Organelles/ultrastructure , Organelles/virology , SARS-CoV-2/physiology
10.
Cells ; 10(3)2021 02 26.
Article in English | MEDLINE | ID: covidwho-1107394

ABSTRACT

Coronaviruses (CoVs) assemble by budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)-Golgi interface. However, why CoVs have chosen the IC as their intracellular site of assembly and how progeny viruses are delivered from this compartment to the extracellular space has remained unclear. Here we address these enigmatic late events of the CoV life cycle in light of recently described properties of the IC. Of particular interest are the emerging spatial and functional connections between IC elements and recycling endosomes (REs), defined by the GTPases Rab1 and Rab11, respectively. The establishment of IC-RE links at the cell periphery, around the centrosome and evidently also at the noncompact zones of the Golgi ribbon indicates that-besides traditional ER-Golgi communication-the IC also promotes a secretory process that bypasses the Golgi stacks, but involves its direct connection with the endocytic recycling system. The initial confinement of CoVs to the lumen of IC-derived large transport carriers and their preferential absence from Golgi stacks is consistent with the idea that they exit cells following such an unconventional route. In fact, CoVs may share this pathway with other intracellularly budding viruses, lipoproteins, procollagen, and/or protein aggregates experimentally introduced into the IC lumen.


Subject(s)
Endoplasmic Reticulum/virology , Extracellular Space/virology , Golgi Apparatus/virology , Intracellular Membranes/virology , SARS-CoV-2/physiology , Secretory Pathway , Virus Release , Animals , COVID-19/therapy , COVID-19/virology , Centrosome/metabolism , Extracellular Space/metabolism , Golgi Apparatus/metabolism , Humans , Protein Transport
11.
Biol Cell ; 113(6): 281-293, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1087985

ABSTRACT

BACKGROUND INFORMATION: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS-CoV-2 isolate from São Paulo state (Brazil). RESULTS: Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. CONCLUSIONS AND SIGNIFICANCE: This study contributes to a better understanding of the cell biology of SARS-CoV-2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus-induced membrane remodelling.


Subject(s)
Endoplasmic Reticulum/virology , Intracellular Membranes/virology , Nuclear Envelope/virology , SARS-CoV-2 , Animals , COVID-19 , Chlorocebus aethiops , Electron Microscope Tomography , Endoplasmic Reticulum/ultrastructure , Humans , Intracellular Membranes/ultrastructure , Microscopy, Electron, Transmission , Nuclear Envelope/ultrastructure , SARS-CoV-2/growth & development , SARS-CoV-2/ultrastructure , Vero Cells , Virus Assembly , Virus Replication
12.
Mem. Inst. Oswaldo Cruz ; 116: e200443, 2021. tab, graf
Article in English | WHO COVID, LILACS (Americas) | ID: covidwho-1076303

ABSTRACT

BACKGROUND The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.


Subject(s)
Humans , Animals , Vero Cells/virology , Cytoplasmic Vesicles/virology , Cytopathogenic Effect, Viral , SARS-CoV-2/physiology , Chlorocebus aethiops , Nucleocapsid , Reverse Transcriptase Polymerase Chain Reaction , Microscopy, Electron, Transmission , Endocytosis , Endoplasmic Reticulum/virology , Virus Internalization , Real-Time Polymerase Chain Reaction
13.
J Biol Chem ; 296: 100111, 2021.
Article in English | MEDLINE | ID: covidwho-1066049

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ß-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of ß-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected versus transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is relocalized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M, and N are required for optimal production of virus-like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.


Subject(s)
Coronavirus Envelope Proteins/genetics , Nucleocapsid Proteins/genetics , SARS-CoV-2/growth & development , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/genetics , Virion/growth & development , Virus Assembly/physiology , Animals , Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Envelope Proteins/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum/virology , Gene Expression , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Golgi Apparatus/virology , HEK293 Cells , Hepatocytes/metabolism , Hepatocytes/ultrastructure , Hepatocytes/virology , Host-Pathogen Interactions/genetics , Humans , Nucleocapsid Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Matrix Proteins/metabolism , Virion/genetics , Virion/metabolism , Virus Internalization , Virus Release/physiology
14.
Virology ; 556: 9-22, 2021 04.
Article in English | MEDLINE | ID: covidwho-985483

ABSTRACT

Coronaviruses rearrange endoplasmic reticulum (ER) membranes to form a reticulovesicular network (RVN) comprised predominantly of double membrane vesicles (DMVs) involved in viral replication. While portions of the RVN have been analyzed by electron tomography (ET), the full extent of the RVN is not known, nor how RVN formation affects ER morphology. Additionally the precise mechanism of DMV formation has not been observed. In this work, we examined large volumes of coronavirus-infected cells at multiple timepoints during infection using serial-section ET. We provide a comprehensive 3D analysis of the ER and RVN which gives insight into the formation mechanism of DMVs as well as the first evidence for their lysosomal degradation. We also show that the RVN breaks down late in infection, concurrent with the ER becoming the main budding compartment for new virions. This work provides a broad view of the multifaceted involvement of ER membranes in coronavirus infection.


Subject(s)
Coronavirus Infections/virology , Endoplasmic Reticulum/metabolism , Murine hepatitis virus/physiology , Viral Replication Compartments/metabolism , Animals , Cell Line , Electron Microscope Tomography , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum/virology , Lysosomes/metabolism , Lysosomes/ultrastructure , Lysosomes/virology , Mice , Viral Proteins/metabolism , Viral Replication Compartments/ultrastructure , Virion/metabolism , Virus Assembly , Virus Replication
15.
Nat Commun ; 11(1): 5885, 2020 11 18.
Article in English | MEDLINE | ID: covidwho-933684

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic ß-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/physiology , Virus Replication , A549 Cells , Animals , COVID-19 , Cell Line , Chlorocebus aethiops , Coronavirus Infections/virology , Cryoelectron Microscopy , Cytoplasmic Vesicles/virology , Electron Microscope Tomography , Endoplasmic Reticulum/virology , Humans , Pandemics , Pneumonia, Viral/virology , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2 , Vero Cells , Virion/chemistry , Virion/metabolism , Virus Assembly
16.
Exp Cell Res ; 396(1): 112276, 2020 11 01.
Article in English | MEDLINE | ID: covidwho-752714

ABSTRACT

Autophagy is an evolutionary conserved catabolic process devoted to the removal of unnecessary and harmful cellular components. In its general form, autophagy governs cellular lifecycle through the formation of double membrane vesicles, termed autophagosomes, that enwrap and deliver unwanted intracellular components to lysosomes. In addition to this omniscient role, forms of selective autophagy, relying on specialized receptors for cargo recognition, exert fine-tuned control over cellular homeostasis. In this regard, xenophagy plays a pivotal role in restricting the replication of intracellular pathogens, thus acting as an ancient innate defense system against infections. Recently, selective autophagy of the endoplasmic reticulum (ER), more simply ER-phagy, has been uncovered as a critical mechanism governing ER network shape and function. Six ER-resident proteins have been characterized as ER-phagy receptors and their orchestrated function enables ER homeostasis and turnover overtime. Unfortunately, ER is also the preferred site for viral replication and several viruses hijack ER machinery for their needs. Thus, it is not surprising that some ER-phagy receptors can act to counteract viral replication and minimize the spread of infection throughout the organism. On the other hand, evolutionary pressure has armed pathogens with strategies to evade and subvert xenophagy and ER-phagy. Although ER-phagy biology is still in its infancy, the present review aims to summarize recent ER-phagy literature, with a special focus on its role in counteracting viral infections. Moreover, we aim to offer some hints for future targeted approaches to counteract host-pathogen interactions by modulating xenophagy and ER-phagy pathways.


Subject(s)
Autophagosomes/immunology , Bacterial Infections/immunology , Endoplasmic Reticulum/immunology , Host-Pathogen Interactions/immunology , Macroautophagy/immunology , Virus Diseases/immunology , Autophagosomes/metabolism , Bacteria/immunology , Bacterial Infections/genetics , Bacterial Infections/microbiology , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/microbiology , Endoplasmic Reticulum/virology , Endoplasmic Reticulum Stress/genetics , Endoplasmic Reticulum Stress/immunology , Homeostasis/genetics , Homeostasis/immunology , Host-Pathogen Interactions/genetics , Humans , Immunity, Innate , Lysosomes/immunology , Lysosomes/metabolism , Macroautophagy/genetics , Virus Diseases/genetics , Virus Diseases/virology , Viruses/immunology
18.
PLoS Biol ; 18(6): e3000715, 2020 06.
Article in English | MEDLINE | ID: covidwho-574821

ABSTRACT

Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.


Subject(s)
Coronavirus Infections/pathology , Coronavirus Infections/virology , Coronavirus/classification , Coronavirus/physiology , Endoplasmic Reticulum/pathology , Endoplasmic Reticulum/virology , Virus Replication , Animals , Betacoronavirus/genetics , Betacoronavirus/physiology , COVID-19 , Cell Line , Chlorocebus aethiops , Electron Microscope Tomography , Endoplasmic Reticulum/ultrastructure , Humans , Middle East Respiratory Syndrome Coronavirus/physiology , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , RNA, Viral/metabolism , SARS-CoV-2 , Vero Cells
19.
Traffic ; 21(8): 545-551, 2020 08.
Article in English | MEDLINE | ID: covidwho-306007

ABSTRACT

Positive sense (+) RNA viruses exploit membranes from a variety of cellular organelles to support the amplification of their genomes. This association concurs with the formation of vesicles whose main morphological feature is that of being wrapped by a double membrane. In the case of the SARS-CoV virus, the outer membrane is not discrete for each vesicle, but seems to be continuous and shared between many individual vesicles, a difference with other +RNA viruses whose nature has remained elusive. I present morphological, biochemical and pharmacological arguments defending the striking analogy of this arrangement and that of entangled, nascent Lipid Droplets whose birth has been aborted by an excess of Phosphatidic Acid. Since Phosphatidic Acid can be targeted with therapeutical purposes, considering this working hypothesis may prove important in tackling SARS-CoV infection.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Models, Biological , Phosphatidic Acids/metabolism , Pneumonia, Viral/metabolism , Pneumonia, Viral/virology , Betacoronavirus/pathogenicity , COVID-19 , Host Microbial Interactions/physiology , Humans , Lipid Droplets/metabolism , Lipid Droplets/virology , Pandemics , SARS-CoV-2 , Virus Replication/physiology
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