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2.
Int J Mol Sci ; 23(4)2022 Feb 09.
Article in English | MEDLINE | ID: covidwho-1690219

ABSTRACT

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2/physiology , Virus Replication/physiology , Coronavirus Nucleocapsid Proteins/analysis , Giant Cells/drug effects , Giant Cells/virology , HEK293 Cells , Humans , Limit of Detection , Nasopharynx/virology , Phosphoproteins/analysis , Phosphoproteins/genetics , RNA, Antisense/pharmacology , RNA, Viral , Ribonuclease P/genetics , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Sensitivity and Specificity , Social Isolation , Viral Load , Viroporin Proteins/genetics , Virus Replication/drug effects
3.
Brief Bioinform ; 23(1)2022 01 17.
Article in English | MEDLINE | ID: covidwho-1597924

ABSTRACT

The pharmacological arsenal against the COVID-19 pandemic is largely based on generic anti-inflammatory strategies or poorly scalable solutions. Moreover, as the ongoing vaccination campaign is rolling slower than wished, affordable and effective therapeutics are needed. To this end, there is increasing attention toward computational methods for drug repositioning and de novo drug design. Here, multiple data-driven computational approaches are systematically integrated to perform a virtual screening and prioritize candidate drugs for the treatment of COVID-19. From the list of prioritized drugs, a subset of representative candidates to test in human cells is selected. Two compounds, 7-hydroxystaurosporine and bafetinib, show synergistic antiviral effects in vitro and strongly inhibit viral-induced syncytia formation. Moreover, since existing drug repositioning methods provide limited usable information for de novo drug design, the relevant chemical substructures of the identified drugs are extracted to provide a chemical vocabulary that may help to design new effective drugs.


Subject(s)
Antiviral Agents/pharmacology , COVID-19 , Giant Cells , Pyrimidines/pharmacology , SARS-CoV-2/metabolism , Staurosporine/analogs & derivatives , A549 Cells , COVID-19/drug therapy , COVID-19/metabolism , Computational Biology , Drug Evaluation, Preclinical , Drug Repositioning , Giant Cells/metabolism , Giant Cells/virology , Humans , Staurosporine/pharmacology
4.
Nature ; 602(7896): 300-306, 2022 02.
Article in English | MEDLINE | ID: covidwho-1532072

ABSTRACT

During the current coronavirus disease 2019 (COVID-19) pandemic, a variety of mutations have accumulated in the viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and, at the time of writing, four variants of concern are considered to be potentially hazardous to human society1. The recently emerged B.1.617.2/Delta variant of concern is closely associated with the COVID-19 surge that occurred in India in the spring of 2021 (ref. 2). However, the virological properties of B.1.617.2/Delta remain unclear. Here we show that the B.1.617.2/Delta variant is highly fusogenic and notably more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates cleavage of the spike protein and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity compared with its parental virus. Our data suggest that the P681R mutation is a hallmark of the virological phenotype of the B.1.617.2/Delta variant and is associated with enhanced pathogenicity.


Subject(s)
COVID-19/virology , Membrane Fusion , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Substitution , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/epidemiology , Cricetinae , Giant Cells/metabolism , Giant Cells/virology , Male , Mesocricetus , Phylogeny , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Virulence/genetics , Virus Replication
5.
Biol Direct ; 16(1): 20, 2021 10 21.
Article in English | MEDLINE | ID: covidwho-1477450

ABSTRACT

SARS-CoV-2 infection could cause severe acute respiratory syndrome, largely attributed to dysregulated immune activation and extensive lung tissue damage. However, the underlying mechanisms are not fully understood. Here, we reported that viral infection could induce syncytia formation within cells expressing ACE2 and the SARS-CoV-2 spike protein, leading to the production of micronuclei with an average rate of about 4 per syncytium (> 93%). Remarkably, these micronuclei were manifested with a high level of activation of both DNA damage response and cGAS-STING signaling, as indicated by micronucleus translocation of γH2Ax and cGAS, and upregulation of their respective downstream target genes. Since activation of these signaling pathways were known to be associated with cellular catastrophe and aberrant immune activation, these findings help explain the pathological effects of SARS-CoV-2 infection at cellular and molecular levels, and provide novel potential targets for COVID-19 therapy.


Subject(s)
COVID-19/genetics , COVID-19/metabolism , DNA Damage , Membrane Proteins/metabolism , Nucleotidyltransferases/metabolism , SARS-CoV-2/pathogenicity , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Giant Cells/metabolism , Giant Cells/virology , HeLa Cells , Humans , Micronucleus Tests , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Signal Transduction , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
6.
EMBO J ; 40(24): e108944, 2021 12 15.
Article in English | MEDLINE | ID: covidwho-1444546

ABSTRACT

Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco-2, Calu-3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon-induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/pharmacology , Giant Cells/virology , Mutation , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Animals , Caco-2 Cells , Cell Line , Chlorocebus aethiops , Giant Cells/drug effects , Giant Cells/metabolism , HEK293 Cells , Humans , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Vero Cells , Virus Replication/drug effects
7.
Immunol Res ; 69(6): 496-519, 2021 12.
Article in English | MEDLINE | ID: covidwho-1363786

ABSTRACT

The SARS-CoV-2 S protein on the membrane of infected cells can promote receptor-dependent syncytia formation, relating to extensive tissue damage and lymphocyte elimination. In this case, it is challenging to obtain neutralizing antibodies and prevent them through antibodies effectively. Considering that, in the current study, structural domain search methods are adopted to analyze the SARS-CoV-2 S protein to find the fusion mechanism. The results show that after the EF-hand domain of S protein bound to calcium ions, S2 protein had CaMKII protein activities. Besides, the CaMKII_AD domain of S2 changed S2 conformation, facilitating the formation of HR1-HR2 six-helix bundles. Apart from that, the Ca2+-ATPase of S2 pumped calcium ions from the virus cytoplasm to help membrane fusion, while motor structures of S drove the CaATP_NAI and CaMKII_AD domains to extend to the outside and combined the viral membrane and the cell membrane, thus forming a calcium bridge. Furthermore, the phospholipid-flipping-ATPase released water, triggering lipid mixing and fusion and generating fusion pores. Then, motor structures promoted fusion pore extension, followed by the cytoplasmic contents of the virus being discharged into the cell cytoplasm. After that, the membrane of the virus slid onto the cell membrane along the flowing membrane on the gap of the three CaATP_NAI. At last, the HR1-HR2 hexamer would fall into the cytoplasm or stay on the cell membrane. Therefore, the CaMKII_like system of S protein facilitated membrane fusion for further inducing syncytial multinucleated giant cells.


Subject(s)
COVID-19/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Transporting ATPases/metabolism , Giant Cells/metabolism , Membrane Fusion/physiology , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Calcium/metabolism , Cell Membrane/physiology , Cell Membrane/virology , Giant Cells/virology , Humans , SARS-CoV-2 , Sequence Alignment , Virus Internalization
8.
Biochem Biophys Res Commun ; 571: 152-158, 2021 09 24.
Article in English | MEDLINE | ID: covidwho-1330656

ABSTRACT

Potent neutralizing antibodies (Abs) have been proven with therapeutic efficacy for the intervention against SARS-CoV-2. Majority of these Abs function by directly interfering with the virus entry to host cells. Here, we identified a receptor binding domain (RBD) specific monoclonal Ab (mAb) 82A6 with efficient neutralizing potency against authentic SARS-CoV-2 virus. As most Abs targeting the non-receptor binding motif (RBM) region, 82A6 was incapable to block the RBD-ACE2 interaction. In particular, it actively promoted the S1 subunit shedding from the S protein, which may lead to effective reduction of intact SARS-CoV-2 viruses. Importantly, it could block potential syncytia formation associated with post-infectious cell surface expression of S proteins. Our study evidenced a RBD specific Ab with unique beneficial efficacy against SARS-CoV-2 infection, which might bring informative significance to understand the collective effects of neutralizing Abs elicited in COVID-19 patients.


Subject(s)
Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , COVID-19/therapy , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Monoclonal/therapeutic use , Antibody Specificity , Binding Sites/immunology , COVID-19/immunology , COVID-19/virology , Giant Cells/immunology , Giant Cells/virology , HEK293 Cells , Humans , Immunization, Passive , In Vitro Techniques , Protein Domains , Protein Subunits , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/chemistry , Virus Shedding
9.
mBio ; 12(4): e0058721, 2021 08 31.
Article in English | MEDLINE | ID: covidwho-1327613

ABSTRACT

Since the D614G substitution in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, the variant strain has undergone a rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage for viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614 (S-G614 and S-D614, respectively). The plaque assay showed a significantly higher virus titer in S-G614 than in S-D614 isolates. We further found increased cleavage of the S protein at the furin substrate site, a key event that promotes syncytium formation, in S-G614 isolates. The enhancement of the D614G substitution in the cleavage of the S protein and in syncytium formation has been validated in cells expressing S protein. The effect on the syncytium was abolished by furin inhibitor treatment and mutation of the furin cleavage site, suggesting its dependence on cleavage by furin. Our study pointed to the impact of the D614G substitution on syncytium formation through enhanced furin-mediated S cleavage, which might increase the transmissibility and infectivity of SARS-CoV-2 strains containing S-G614. IMPORTANCE Analysis of viral genomes and monitoring of the evolutionary trajectory of SARS-CoV-2 over time has identified the D614G substitution in spike (S) as the most prevalent expanding variant worldwide, which might confer a selective advantage in transmission. Several studies showed that the D614G variant replicates and transmits more efficiently than the wild-type virus, but the mechanism is unclear. By comparing 18 virus isolates containing S with either D614 or G614, we found significantly higher virus titers in association with higher furin protease-mediated cleavage of S, an event that promotes syncytium formation and virus infectivity, in the S-G614 viruses. The effect of the D614G substitution on furin-mediated S cleavage and the resulting enhancement of the syncytium phenotype has been validated in S-expressing cells. This study suggests a possible effect of the D614G substitution on S of SARS-CoV-2; the antiviral effect through targeting furin protease is worthy of being investigated in proper animal models.


Subject(s)
COVID-19/transmission , Furin/metabolism , Giant Cells/virology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Substitution/genetics , Animals , COVID-19/pathology , Cell Line , Chlorocebus aethiops , Furin/antagonists & inhibitors , Genetic Fitness/genetics , Genome, Viral/genetics , HEK293 Cells , Humans , SARS-CoV-2/isolation & purification , Vero Cells , Viral Load/genetics , Virus Replication/genetics
10.
mBio ; 12(3): e0078821, 2021 06 29.
Article in English | MEDLINE | ID: covidwho-1286718

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus that is continuously evolving. Although its RNA-dependent RNA polymerase exhibits some exonuclease proofreading activity, viral sequence diversity can be produced by replication errors and host factors. A diversity of genetic variants can be observed in the intrahost viral population structure of infected individuals. Most mutations will follow a neutral molecular evolution and will not make significant contributions to variations within and between infected hosts. Herein, we profiled the intrasample genetic diversity of SARS-CoV-2 variants, also known as quasispecies, using high-throughput sequencing data sets from 15,289 infected individuals and infected cell lines. Despite high mutational background, we identified recurrent intragenetic variable positions in the samples analyzed, including several positions at the end of the gene encoding the viral spike (S) protein. Strikingly, we observed a high frequency of C→A missense mutations resulting in the S protein lacking the last 20 amino acids (SΔ20). We found that this truncated S protein undergoes increased processing and increased syncytium formation, presumably due to escaping M protein retention in intracellular compartments. Our findings suggest the emergence of a high-frequency viral sublineage that is not horizontally transmitted but potentially involved in intrahost disease cytopathic effects. IMPORTANCE The mutation rate and evolution of RNA viruses correlate with viral adaptation. While most mutations do not make significant contributions to viral molecular evolution, some are naturally selected and produce variants through positive selection. Many SARS-CoV-2 variants have been recently described and show phenotypic selection toward more infectious viruses. Our study describes another type of variant that does not contribute to interhost heterogeneity but rather phenotypic selection toward variants that might have increased cytopathic effects. We identified that a C-terminal truncation of the spike protein removes an important endoplasmic reticulum (ER) retention signal, which consequently results in a spike variant that easily travels through the Golgi complex toward the plasma membrane in a preactivated conformation, leading to increased syncytium formation.


Subject(s)
COVID-19/pathology , Genome, Viral/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Cell Line , Evolution, Molecular , Genetic Variation/genetics , Giant Cells/virology , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , Mutation Rate , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology
11.
Sci Rep ; 11(1): 9136, 2021 04 28.
Article in English | MEDLINE | ID: covidwho-1207152

ABSTRACT

Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism of viral spike protein-mediated fusion or screening for viral inhibitors under biosafety level 1 conditions.


Subject(s)
Host-Pathogen Interactions/physiology , Peptides/chemistry , Peptides/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Cell Fusion , Circular Dichroism , Giant Cells/virology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Membrane Fusion , Peptides/genetics , Protein Engineering/methods , Protein Multimerization , Protein Stability , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Transcription, Genetic
12.
Cell Death Differ ; 28(9): 2765-2777, 2021 09.
Article in English | MEDLINE | ID: covidwho-1195611

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is highly contagious and causes lymphocytopenia, but the underlying mechanisms are poorly understood. We demonstrate here that heterotypic cell-in-cell structures with lymphocytes inside multinucleate syncytia are prevalent in the lung tissues of coronavirus disease 2019 (COVID-19) patients. These unique cellular structures are a direct result of SARS-CoV-2 infection, as the expression of the SARS-CoV-2 spike glycoprotein is sufficient to induce a rapid (~45.1 nm/s) membrane fusion to produce syncytium, which could readily internalize multiple lines of lymphocytes to form typical cell-in-cell structures, remarkably leading to the death of internalized cells. This membrane fusion is dictated by a bi-arginine motif within the polybasic S1/S2 cleavage site, which is frequently present in the surface glycoprotein of most highly contagious viruses. Moreover, candidate anti-viral drugs could efficiently inhibit spike glycoprotein processing, membrane fusion, and cell-in-cell formation. Together, we delineate a molecular and cellular rationale for SARS-CoV-2 pathogenesis and identify novel targets for COVID-19 therapy.


Subject(s)
COVID-19/virology , Giant Cells/virology , Lymphocytes/virology , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , COVID-19/pathology , Cell Line , Cell Line, Tumor , Giant Cells/pathology , HEK293 Cells , HeLa Cells , Humans , Jurkat Cells , K562 Cells , Lymphocytes/pathology , Virus Internalization , Virus Replication/genetics
13.
Nature ; 594(7861): 88-93, 2021 06.
Article in English | MEDLINE | ID: covidwho-1171428

ABSTRACT

COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin 6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.


Subject(s)
Anoctamins/antagonists & inhibitors , COVID-19/pathology , Cell Fusion , Drug Evaluation, Preclinical , Giant Cells/drug effects , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Aged , Aged, 80 and over , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Animals , Anoctamins/metabolism , COVID-19/metabolism , COVID-19/virology , Calcium Signaling/drug effects , Cell Line , Chloride Channels/metabolism , Chlorocebus aethiops , Female , Giant Cells/metabolism , Giant Cells/virology , Humans , Lung/drug effects , Lung/pathology , Lung/virology , Male , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Virus Replication/drug effects
14.
EBioMedicine ; 61: 103104, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-912159

ABSTRACT

BACKGROUND: COVID-19 is a deadly pulmonary disease with peculiar characteristics, which include variable clinical course and thrombophilia. A thorough understanding of the pathological correlates of the disease is still missing. METHODS: Here we report the systematic analysis of 41 consecutive post-mortem samples from individuals who died of COVID-19. Histological analysis is complemented by immunohistochemistry for cellular and viral antigens and the detection of viral genomes by in situ RNA hybridization. FINDINGS: COVID-19 is characterized by extensive alveolar damage (41/41 of patients) and thrombosis of the lung micro- and macro-vasculature (29/41, 71%). Thrombi were in different stages of organization, consistent with their local origin. Pneumocytes and endothelial cells contained viral RNA even at the later stages of the disease. An additional feature was the common presence of a large number of dysmorphic pneumocytes, often forming syncytial elements (36/41, 87%). Despite occasional detection of virus-positive cells, no overt signs of viral infection were detected in other organs, which showed non-specific alterations. INTERPRETATION: COVID-19 is a unique disease characterized by extensive lung thrombosis, long-term persistence of viral RNA in pneumocytes and endothelial cells, along with the presence of infected cell syncytia. Several of COVID-19 features might be consequent to the persistence of virus-infected cells for the duration of the disease. FUNDING: This work was supported by a King's Together Rapid COVID-19 Call grant from King's College London. MG is supported by the European Research Council (ERC) Advanced Grant 787971 "CuRE" and by Programme Grant RG/19/11/34633 from the British Heart Foundation.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/pathology , Pneumonia, Viral/pathology , RNA, Viral/metabolism , Thrombosis/etiology , Aged , Aged, 80 and over , Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/virology , Autopsy , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/complications , Coronavirus Infections/virology , Critical Care , Endothelial Cells/virology , Female , Giant Cells/cytology , Giant Cells/virology , Humans , Lung/pathology , Lung/virology , Male , Pandemics , Pneumonia, Viral/complications , Pneumonia, Viral/virology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism
15.
J Med Virol ; 92(10): 2087-2095, 2020 10.
Article in English | MEDLINE | ID: covidwho-763177

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) is the causative agent of the coronavirus disease-2019 (COVID-19) pandemic. Coronaviruses enter cells via fusion of the viral envelope with the plasma membrane and/or via fusion of the viral envelope with endosomal membranes after virion endocytosis. The spike (S) glycoprotein is a major determinant of virus infectivity. Herein, we show that the transient expression of the SARS CoV-2 S glycoprotein in Vero cells caused extensive cell fusion (formation of syncytia) in comparison to limited cell fusion caused by the SARS S glycoprotein. Both S glycoproteins were detected intracellularly and on transfected Vero cell surfaces. These results are in agreement with published pathology observations of extensive syncytia formation in lung tissues of patients with COVID-19. These results suggest that SARS CoV-2 is able to spread from cell-to-cell much more efficiently than SARS effectively avoiding extracellular neutralizing antibodies. A systematic screening of several drugs including cardiac glycosides and kinase inhibitors and inhibitors of human immunodeficiency virus (HIV) entry revealed that only the FDA-approved HIV protease inhibitor, nelfinavir mesylate (Viracept) drastically inhibited S-n- and S-o-mediated cell fusion with complete inhibition at a 10-µM concentration. In-silico docking experiments suggested the possibility that nelfinavir may bind inside the S trimer structure, proximal to the S2 amino terminus directly inhibiting S-n- and S-o-mediated membrane fusion. Also, it is possible that nelfinavir may act to inhibit S proteolytic processing within cells. These results warrant further investigations of the potential of nelfinavir mesylate to inhibit virus spread at early times after SARS CoV-2 symptoms appear.


Subject(s)
Anti-HIV Agents/pharmacology , Membrane Fusion/drug effects , Nelfinavir/pharmacology , SARS Virus/drug effects , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Animals , Anti-HIV Agents/chemistry , Binding Sites , COVID-19/drug therapy , Cell Fusion , Chlorocebus aethiops , Giant Cells/drug effects , Giant Cells/pathology , Giant Cells/virology , Humans , Molecular Docking Simulation , Nelfinavir/chemistry , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS Virus/pathogenicity , SARS Virus/physiology , SARS-CoV-2/pathogenicity , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virion/drug effects , Virion/pathogenicity , Virion/physiology
16.
Placenta ; 97: 1-5, 2020 08.
Article in English | MEDLINE | ID: covidwho-437293

ABSTRACT

Although many pregnant women have been infected by coronavirus, the presence of intrauterine vertical transmission has not been conclusively reported yet. What prevents this highly contagious virus from reaching the fetus? Is it only the presence of a strong placental barrier, or is it the natural absence of the some receptor that the viruses use for transmission? We, therefore, need to comprehensively understand the mechanism of action of the mammalian epithelial barriers located in two different organs with functional similarity. The barriers selected as potential targets by SARS-CoV-2 are the alveolo-capillary barrier (ACB), and the syncytio-capillary barrier (SCB). Caveolae are omega-shaped structures located on the cell membrane. They consist of caveolin-1 protein (Cav-1) and are involved in the internalisation of some viruses. By activating leukocytes and nuclear factor-κB, Cav-1 initiates inflammatory reactions. The presence of more than one Cav-1 binding sites on coronavirus is an important finding supporting the possible relationship between SARS-CoV-2-mediated lung injury. While the ACB cells express Cav-1 there is no caveolin expression in syncytiotrophoblasts. In this short review, we will try to explain our hypothesis that the lack of caveolin expression in the SCB is one of the most important physiological mechanisms that prevents vertical transmission of SARS-CoV-2. Since the physiological Cav-1 deficiency appears to prevent acute cell damage treatment algorithms could potentially be developed to block this pathway in the non-pregnant population affected by SARS-CoV-2.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/prevention & control , Coronavirus Infections/transmission , Fetal Diseases/prevention & control , Infectious Disease Transmission, Vertical/prevention & control , Maternal-Fetal Exchange/immunology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Pneumonia, Viral/transmission , Betacoronavirus/immunology , COVID-19 , Caveolin 1/physiology , Coronavirus Infections/immunology , Epithelium/physiology , Epithelium/virology , Female , Fetal Diseases/immunology , Fetal Diseases/virology , Giant Cells/physiology , Giant Cells/virology , Humans , Immunity, Innate/physiology , Pneumonia, Viral/immunology , Pregnancy , Risk Factors , SARS-CoV-2 , Virus Internalization
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