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1.
Microbiol Spectr ; 10(1): e0127121, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1752773

ABSTRACT

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the coronavirus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS-CoV-2-infected Vero cells, processed identically. Correlative light and electron microscopy on semithick cryo-sections demonstrated induction of electron-lucent, lipid-filled compartments after SARS-CoV-2 infection in both lung and cell cultures. In lung tissue, the nonstructural protein 4 and the stable nucleocapsid N-protein were detected on these novel lipid-filled compartments. The induction of such lipid-filled compartments and the localization of the viral proteins in lung of patients with fatal COVID-19 may explain the extensive inflammatory response and provide a new hallmark for SARS-CoV-2 infection at the final, fatal stage of infection. IMPORTANCE Visualization of the subcellular localization of SARS-CoV-2 proteins in lung patient material of COVID-19 patients is important for the understanding of this new virus. We detected viral proteins in the context of the ultrastructure of infected cells and tissues and discovered that some viral proteins accumulate in novel, lipid-filled compartments. These structures are induced in Vero cells but, more importantly, also in lung of patients with COVID-19. We have characterized these lipid-filled compartments and determined that this is a novel, virus-induced structure. Immunogold labeling demonstrated that cellular markers, such as CD63 and lipid droplet marker PLIN-2, are absent. Colocalization of lipid-filled compartments with the stable N-protein and nonstructural protein 4 in lung of the last stages of COVID-19 indicates that these compartments play a key role in the devastating immune response that SARS-CoV-2 infections provoke.


Subject(s)
COVID-19/metabolism , Lipid Metabolism/physiology , Lipids/analysis , Lung/metabolism , Nucleocapsid/analysis , SARS-CoV-2 , Adolescent , Aged , Animals , COVID-19/pathology , Child, Preschool , Chlorocebus aethiops , Disease Outbreaks , Female , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Lung/cytology , Lung/pathology , Lung/ultrastructure , Male , Microscopy, Immunoelectron , Middle Aged , Nucleocapsid/metabolism , Rabbits , SARS-CoV-2/ultrastructure , Vero Cells/virology
2.
Nat Commun ; 13(1): 1214, 2022 03 03.
Article in English | MEDLINE | ID: covidwho-1730288

ABSTRACT

The omicron variant of SARS-CoV-2 has been spreading rapidly across the globe. The virus-surface spike protein plays a critical role in the cell entry and immune evasion of SARS-CoV-2. Here we determined the 3.0 Å cryo-EM structure of the omicron spike protein ectodomain. In contrast to the original strain of SARS-CoV-2 where the receptor-binding domain (RBD) of the spike protein takes a mixture of open ("standing up") and closed ("lying down") conformations, the omicron spike molecules are predominantly in the open conformation, with one upright RBD ready for receptor binding. The open conformation of the omicron spike is stabilized by enhanced inter-domain and inter-subunit packing, which involves new mutations in the omicron strain. Moreover, the omicron spike has undergone extensive mutations in RBD regions where known neutralizing antibodies target, allowing the omicron variant to escape immune surveillance aimed at the original viral strain. The stable open conformation of the omicron spike sheds light on the cell entry and immune evasion mechanisms of the omicron variant.


Subject(s)
COVID-19/virology , SARS-CoV-2/chemistry , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/ultrastructure , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Cryoelectron Microscopy , Humans , Immune Evasion/genetics , Models, Molecular , Mutation , Pandemics , Protein Conformation , Protein Domains/genetics , Protein Domains/immunology , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization
3.
Med Mol Morphol ; 55(1): 60-67, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1712248

ABSTRACT

SARS-CoV-2 is the cause of COVID-19. The three-dimensional morphology of viral particles existing and multiplying in infected cells has not been established by electron tomography, which is different from cryo-electron tomography using frozen samples. In this study, we establish the morphological structure of SARS-CoV-2 particles by three-dimensional reconstruction of images obtained by electron tomography and transmission electron microscopy of biological samples embedded in epoxy resin. The characteristic roots of spike structures were found to be arranged at the surface of a virion covered with an envelope. A high-electron-density structure that appears to be a nucleocapsid was observed inside the envelope of the virion on three-dimensional images reconstructed by electron tomography. The SARS-CoV-2 particles that budded in the vacuoles in the cytoplasm were morphologically identical to those found outside the cells, suggesting that mature and infectious SARS-CoV-2 particles were already produced in the vacuoles. Here, we show the three-dimensional morphological structure of SARS-CoV-2 particles reconstructed by electron tomography. To control infection, inhibition of viral release from vacuoles would be a new target in the development of prophylactic agents against SARS-CoV-2.


Subject(s)
Electron Microscope Tomography , SARS-CoV-2 , COVID-19 , Humans , Imaging, Three-Dimensional , SARS-CoV-2/ultrastructure , Virion/ultrastructure
5.
Cell Rep ; 38(9): 110428, 2022 03 01.
Article in English | MEDLINE | ID: covidwho-1670282

ABSTRACT

The recently reported B.1.1.529 Omicron variant of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) includes 34 mutations in the spike protein relative to the Wuhan strain, including 15 mutations in the receptor-binding domain (RBD). Functional studies have shown Omicron to substantially escape the activity of many SARS-CoV-2-neutralizing antibodies. Here, we report a 3.1 Å-resolution cryoelectron microscopy (cryo-EM) structure of the Omicron spike protein ectodomain. The structure depicts a spike that is exclusively in the 1-RBD-up conformation with high mobility of RBD. Many mutations cause steric clashes and/or altered interactions at antibody-binding surfaces, whereas others mediate changes of the spike structure in local regions to interfere with antibody recognition. Overall, the structure of the Omicron spike reveals how mutations alter its conformation and explains its extraordinary ability to evade neutralizing antibodies.


Subject(s)
Cryoelectron Microscopy , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/metabolism , Humans , Immune Evasion/genetics , Models, Molecular , Mutation , Neutralization Tests , Protein Binding , Protein Structure, Quaternary , SARS-CoV-2/genetics , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/genetics
6.
Int J Mol Sci ; 23(3)2022 Jan 31.
Article in English | MEDLINE | ID: covidwho-1667196

ABSTRACT

Structural and functional studies of the SARS-CoV-2 spike proteins have recently determined distinct functional states of the B.1.1.7 and B.1.351 spike variants, providing a molecular framework for understanding the mechanisms that link the effect of mutations with the enhanced virus infectivity and transmissibility. A detailed dynamic and energetic analysis of these variants was undertaken in the present work to quantify the effects of different mutations on functional conformational changes and stability of the SARS-CoV-2 spike protein. We employed the efficient and accurate coarse-grained (CG) simulations of multiple functional states of the D614G mutant, B.1.1.7 and B.1.351 spike variants to characterize conformational dynamics of the SARS-CoV-2 spike proteins and identify dynamic signatures of the functional regions that regulate transitions between the closed and open forms. By combining molecular simulations with full atomistic reconstruction of the trajectories and the ensemble-based mutational frustration analysis, we characterized how the intrinsic flexibility of specific spike regions can control functional conformational changes required for binding with the host-cell receptor. Using the residue-based mutational scanning of protein stability, we determined protein stability hotspots and identified potential energetic drivers favoring the receptor-accessible open spike states for the B.1.1.7 and B.1.351 spike variants. The results suggested that modulation of the energetic frustration at the inter-protomer interfaces can serve as a mechanism for allosteric couplings between mutational sites and the inter-protomer hinges of functional motions. The proposed mechanism of mutation-induced energetic frustration may result in greater adaptability and the emergence of multiple conformational states in the open form. This study suggested that SARS-CoV-2 B.1.1.7 and B.1.351 variants may leverage the intrinsic plasticity of functional regions in the spike protein for mutation-induced modulation of protein dynamics and allosteric regulation to control binding with the host cell receptor.


Subject(s)
COVID-19/metabolism , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/ultrastructure , Allosteric Regulation , Binding Sites , COVID-19/pathology , Humans , Molecular Conformation , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Stability , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics
7.
Viruses ; 14(2)2022 01 20.
Article in English | MEDLINE | ID: covidwho-1650717

ABSTRACT

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted public health and the world economy and fueled a worldwide race to approve therapeutic and prophylactic agents, but so far there are no specific antiviral drugs. Understanding the biology of the virus is the first step in structuring strategies to combat it, and in this context several studies have been conducted with the aim of understanding the replication mechanism of SARS-CoV-2 in vitro systems. In this work, studies using transmission and scanning electron microscopy and 3D electron microscopy modeling were performed with the goal of characterizing the morphogenesis of SARS-CoV-2 in Vero-E6 cells. Several ultrastructural changes were observed-such as syncytia formation, cytoplasmic membrane projections, lipid droplets accumulation, proliferation of double-membrane vesicles derived from the rough endoplasmic reticulum, and alteration of mitochondria. The entry of the virus into cells occurred through endocytosis. Viral particles were observed attached to the cell membrane and in various cellular compartments, and extrusion of viral progeny took place by exocytosis. These findings allow us to infer that Vero-E6 cells are highly susceptible to SARS-CoV-2 infection as described in the literature and their replication cycle is similar to that described with SARS-CoV and MERS-CoV in vitro models.


Subject(s)
Microscopy, Electron, Transmission/methods , Microscopy, Electron/methods , SARS-CoV-2/metabolism , SARS-CoV-2/ultrastructure , Animals , Cell Line , Chlorocebus aethiops , SARS-CoV-2/chemistry , Vero Cells , Virus Internalization , Virus Replication
8.
Molecules ; 27(3)2022 Jan 20.
Article in English | MEDLINE | ID: covidwho-1648332

ABSTRACT

In spite of advances in vaccination, control of the COVID-19 pandemic will require the use of pharmacological treatments against SARS-CoV2. Their development needs to consider the existence of two phases in the disease, namely the viral infection and the inflammatory stages. The main targets for antiviral therapeutic intervention are: (a) viral proteins, including the spike (S) protein characteristic of the viral cover and the viral proteases in charge of processing the polyprotein arising from viral genome translation; (b) host proteins, such as those involved in the processes related to viral entry into the host cell and the release of the viral genome inside the cell, the elongation factor eEF1A and importins. The use of antivirals targeted at host proteins is less developed but it has the potential advantage of not being affected by mutations in the genome of the virus and therefore being active against all its variants. Regarding drugs that address the hyperinflammatory phase of the disease triggered by the so-called cytokine storm, the following strategies are particularly relevant: (a) drugs targeting JAK kinases; (b) sphingosine kinase 2 inhibitors; (c) antibodies against interleukin 6 or its receptor; (d) use of the traditional anti-inflammatory corticosteroids.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/pathology , Chemistry, Pharmaceutical , Humans , Inflammation/drug therapy , Inflammation/etiology , SARS-CoV-2/drug effects , SARS-CoV-2/ultrastructure
9.
J Cell Mol Med ; 26(1): 25-34, 2022 01.
Article in English | MEDLINE | ID: covidwho-1570773

ABSTRACT

Transmission electron microscopy has historically been indispensable for virology research, as it offers unique insight into virus function. In the past decade, as cryo-electron microscopy (cryo-EM) has matured and become more accessible, we have been able to peer into the structure of viruses at the atomic level and understand how they interact with the host cell, with drugs or with antibodies. Perhaps, there was no time in recent history where cryo-EM was more needed, as SARS-CoV-2 has spread around the globe, causing millions of deaths and almost unquantifiable economic devastation. In this concise review, we aim to mark the most important contributions of cryo-EM to understanding the structure and function of SARS-CoV-2 proteins, from surface spikes to the virus core and from virus-receptor interactions to antibody binding.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Viral/chemistry , COVID-19 Vaccines/chemistry , COVID-19/prevention & control , Receptors, Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/biosynthesis , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/biosynthesis , Cryoelectron Microscopy , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Humans , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , SARS-CoV-2/ultrastructure , Serine Endopeptidases/chemistry , Serine Endopeptidases/immunology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virion/drug effects , Virion/pathogenicity , Virion/ultrastructure
10.
Med Mol Morphol ; 55(1): 60-67, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1536314

ABSTRACT

SARS-CoV-2 is the cause of COVID-19. The three-dimensional morphology of viral particles existing and multiplying in infected cells has not been established by electron tomography, which is different from cryo-electron tomography using frozen samples. In this study, we establish the morphological structure of SARS-CoV-2 particles by three-dimensional reconstruction of images obtained by electron tomography and transmission electron microscopy of biological samples embedded in epoxy resin. The characteristic roots of spike structures were found to be arranged at the surface of a virion covered with an envelope. A high-electron-density structure that appears to be a nucleocapsid was observed inside the envelope of the virion on three-dimensional images reconstructed by electron tomography. The SARS-CoV-2 particles that budded in the vacuoles in the cytoplasm were morphologically identical to those found outside the cells, suggesting that mature and infectious SARS-CoV-2 particles were already produced in the vacuoles. Here, we show the three-dimensional morphological structure of SARS-CoV-2 particles reconstructed by electron tomography. To control infection, inhibition of viral release from vacuoles would be a new target in the development of prophylactic agents against SARS-CoV-2.


Subject(s)
Electron Microscope Tomography , SARS-CoV-2 , COVID-19 , Humans , Imaging, Three-Dimensional , SARS-CoV-2/ultrastructure , Virion/ultrastructure
11.
Proc Natl Acad Sci U S A ; 118(48)2021 11 30.
Article in English | MEDLINE | ID: covidwho-1517667

ABSTRACT

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of the viral genome into host cells. To understand its detailed entry mechanism and develop a specific entry inhibitor, in situ structural information on the SARS-CoV-2 spike protein in different states is urgent. Here, by using cryo-electron tomography, we observed both prefusion and postfusion spikes in ß-propiolactone-inactivated SARS-CoV-2 virions and solved the in situ structure of the postfusion spike at nanometer resolution. Compared to previous reports, the six-helix bundle fusion core, the glycosylation sites, and the location of the transmembrane domain were clearly resolved. We observed oligomerization patterns of the spikes on the viral membrane, likely suggesting a mechanism of fusion pore formation.


Subject(s)
SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Motifs , Animals , Chlorocebus aethiops , Cryoelectron Microscopy , Electron Microscope Tomography , Glycosylation , Protein Domains , Protein Multimerization , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
12.
Virol J ; 18(1): 149, 2021 07 18.
Article in English | MEDLINE | ID: covidwho-1496197

ABSTRACT

BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.


Subject(s)
COVID-19/pathology , Nasopharynx/ultrastructure , SARS-CoV-2/ultrastructure , Antigens, Viral/metabolism , COVID-19/diagnosis , COVID-19/virology , Epithelial Cells/ultrastructure , Epithelial Cells/virology , Humans , Image Enhancement , Microscopy , Microvilli/ultrastructure , Nasal Mucosa/ultrastructure , Nasal Mucosa/virology , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Virion/ultrastructure
13.
Sci Rep ; 11(1): 18851, 2021 09 22.
Article in English | MEDLINE | ID: covidwho-1434149

ABSTRACT

In this pandemic SARS-CoV-2 crisis, any attempt to contain and eliminate the virus will also stop its spread and consequently decrease the risk of severe illness and death. While ozone treatment has been suggested as an effective disinfection process, no precise mechanism of action has been previously reported. This study aimed to further investigate the effect of ozone treatment on SARS-CoV-2. Therefore, virus collected from nasopharyngeal and oropharyngeal swab and sputum samples from symptomatic patients was exposed to ozone for different exposure times. The virus morphology and structure were monitored and analyzed through Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Atomic Absorption Spectroscopy (AAS), and ATR-FTIR. The obtained results showed that ozone treatment not only unsettles the virus morphology but also alters the virus proteins' structure and conformation through amino acid disturbance and Zn ion release from the virus non-structural proteins. These results could provide a clearer pathway for virus elimination and therapeutics preparation.


Subject(s)
COVID-19/drug therapy , Ozone/pharmacology , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans , Microscopy, Electron, Transmission , Protein Structure, Secondary/drug effects , Protein Structure, Tertiary/drug effects , SARS-CoV-2/ultrastructure , Time Factors , Viral Envelope/chemistry , Viral Envelope/drug effects , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Zinc/chemistry , Zinc/metabolism
14.
Sci Rep ; 11(1): 17748, 2021 09 07.
Article in English | MEDLINE | ID: covidwho-1412634

ABSTRACT

Based on WHO reports the new SARS-CoV-2 coronavirus is currently widespread all over the world. So far > 162 million cases have been confirmed, including > 3 million deaths. Because of the pandemic still spreading across the globe the accomplishment of computational methods to find new potential mechanisms of virus inhibitions is necessary. According to the fact that C60 fullerene (a sphere-shaped molecule consisting of carbon) has shown inhibitory activity against various protein targets, here the analysis of the potential binding mechanism between SARS-CoV-2 proteins 3CLpro and RdRp with C60 fullerene was done; it has resulted in one and two possible binding mechanisms, respectively. In the case of 3CLpro, C60 fullerene interacts in the catalytic binding pocket. And for RdRp in the first model C60 fullerene blocks RNA synthesis pore and in the second one it prevents binding with Nsp8 co-factor (without this complex formation, RdRp can't perform its initial functions). Then the molecular dynamics simulation confirmed the stability of created complexes. The obtained results might be a basis for other computational studies of 3CLPro and RdRp potential inhibition ways as well as the potential usage of C60 fullerene in the fight against COVID-19 disease.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Fullerenes/pharmacology , Antiviral Agents/therapeutic use , COVID-19/epidemiology , COVID-19/virology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/ultrastructure , Coronavirus Protease Inhibitors/chemistry , Coronavirus Protease Inhibitors/pharmacology , Coronavirus Protease Inhibitors/therapeutic use , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/ultrastructure , Crystallography, X-Ray , Fullerenes/chemistry , Fullerenes/therapeutic use , Humans , Molecular Dynamics Simulation , Nucleic Acid Synthesis Inhibitors/chemistry , Nucleic Acid Synthesis Inhibitors/pharmacology , Nucleic Acid Synthesis Inhibitors/therapeutic use , Pandemics/prevention & control , RNA, Viral/biosynthesis , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , SARS-CoV-2/ultrastructure
15.
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
16.
Viruses ; 13(9)2021 09 13.
Article in English | MEDLINE | ID: covidwho-1411078

ABSTRACT

BACKGROUND: There is increasing evidence that identification of SARS-CoV-2 virions by transmission electron microscopy could be misleading due to the similar morphology of virions and ubiquitous cell structures. This study thus aimed to establish methods for indisputable proof of the presence of SARS-CoV-2 virions in the observed tissue. METHODS: We developed a variant of the correlative microscopy approach for SARS-CoV-2 protein identification using immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels. We also performed immunogold labelling of SARS-CoV-2 virions. RESULTS: Immunohistochemistry (IHC) of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of SARS-CoV-2 virions in the analysed human nasopharyngeal tissue. The presence of SARS-CoV-2 virions was also confirmed by immunogold labelling for the first time. CONCLUSIONS: Immunoelectron microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections. Both methodological approaches indisputably proved the presence of SARS-CoV-2 virions in cells.


Subject(s)
COVID-19/virology , SARS-CoV-2/isolation & purification , Virion/isolation & purification , Coronavirus Nucleocapsid Proteins/analysis , Humans , Immunohistochemistry , Microscopy, Immunoelectron , Nasopharynx/virology , Phosphoproteins/analysis , SARS-CoV-2/ultrastructure , Virion/ultrastructure
17.
Sci Rep ; 11(1): 17748, 2021 09 07.
Article in English | MEDLINE | ID: covidwho-1397901

ABSTRACT

Based on WHO reports the new SARS-CoV-2 coronavirus is currently widespread all over the world. So far > 162 million cases have been confirmed, including > 3 million deaths. Because of the pandemic still spreading across the globe the accomplishment of computational methods to find new potential mechanisms of virus inhibitions is necessary. According to the fact that C60 fullerene (a sphere-shaped molecule consisting of carbon) has shown inhibitory activity against various protein targets, here the analysis of the potential binding mechanism between SARS-CoV-2 proteins 3CLpro and RdRp with C60 fullerene was done; it has resulted in one and two possible binding mechanisms, respectively. In the case of 3CLpro, C60 fullerene interacts in the catalytic binding pocket. And for RdRp in the first model C60 fullerene blocks RNA synthesis pore and in the second one it prevents binding with Nsp8 co-factor (without this complex formation, RdRp can't perform its initial functions). Then the molecular dynamics simulation confirmed the stability of created complexes. The obtained results might be a basis for other computational studies of 3CLPro and RdRp potential inhibition ways as well as the potential usage of C60 fullerene in the fight against COVID-19 disease.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Fullerenes/pharmacology , Antiviral Agents/therapeutic use , COVID-19/epidemiology , COVID-19/virology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/ultrastructure , Coronavirus Protease Inhibitors/chemistry , Coronavirus Protease Inhibitors/pharmacology , Coronavirus Protease Inhibitors/therapeutic use , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/ultrastructure , Crystallography, X-Ray , Fullerenes/chemistry , Fullerenes/therapeutic use , Humans , Molecular Dynamics Simulation , Nucleic Acid Synthesis Inhibitors/chemistry , Nucleic Acid Synthesis Inhibitors/pharmacology , Nucleic Acid Synthesis Inhibitors/therapeutic use , Pandemics/prevention & control , RNA, Viral/biosynthesis , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , SARS-CoV-2/ultrastructure
18.
Viruses ; 13(9)2021 09 01.
Article in English | MEDLINE | ID: covidwho-1390787

ABSTRACT

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.


Subject(s)
COVID-19/virology , Host-Pathogen Interactions , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Physiological Phenomena/drug effects , Virus Replication/drug effects , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/metabolism , Cell Cycle , Chlorocebus aethiops , Doxycycline/pharmacology , HEK293 Cells , Humans , Protein Binding , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus , Transduction, Genetic , Vero Cells
19.
Biochemistry ; 60(27): 2153-2169, 2021 07 13.
Article in English | MEDLINE | ID: covidwho-1387101

ABSTRACT

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.


Subject(s)
COVID-19/genetics , Protein Conformation , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/ultrastructure , Animals , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Glycosylation , Humans , Molecular Dynamics Simulation , Protein Binding/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
20.
Commun Biol ; 4(1): 874, 2021 07 15.
Article in English | MEDLINE | ID: covidwho-1387496

ABSTRACT

Cryo-EM maps are valuable sources of information for protein structure modeling. However, due to the loss of contrast at high frequencies, they generally need to be post-processed to improve their interpretability. Most popular approaches, based on global B-factor correction, suffer from limitations. For instance, they ignore the heterogeneity in the map local quality that reconstructions tend to exhibit. Aiming to overcome these problems, we present DeepEMhancer, a deep learning approach designed to perform automatic post-processing of cryo-EM maps. Trained on a dataset of pairs of experimental maps and maps sharpened using their respective atomic models, DeepEMhancer has learned how to post-process experimental maps performing masking-like and sharpening-like operations in a single step. DeepEMhancer was evaluated on a testing set of 20 different experimental maps, showing its ability to reduce noise levels and obtain more detailed versions of the experimental maps. Additionally, we illustrated the benefits of DeepEMhancer on the structure of the SARS-CoV-2 RNA polymerase.


Subject(s)
Cryoelectron Microscopy/instrumentation , DNA-Directed RNA Polymerases/ultrastructure , Deep Learning , SARS-CoV-2/ultrastructure , Viral Proteins/ultrastructure
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