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1.
Anal Bioanal Chem ; 414(5): 1773-1785, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1653430

ABSTRACT

Nucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 105 to 2.0 × 105 copies/mL. The RM has a between-bottle homogeneity of 4.80-8.23% and is stable at 4 °C for 1 week and at -70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Genome, Viral , RNA, Viral/genetics , Reagent Kits, Diagnostic/standards , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Gene Dosage , Gene Expression , Humans , Jurkat Cells , Lentivirus/genetics , Lentivirus/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , RNA, Viral/metabolism , RNA, Viral/standards , Reagent Kits, Diagnostic/supply & distribution , Reference Standards , Reproducibility of Results , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Genome Packaging
2.
Proc Natl Acad Sci U S A ; 119(5)2022 02 01.
Article in English | MEDLINE | ID: covidwho-1642084

ABSTRACT

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between levels of viral RNA and infectious virus for individual variants is unknown. We measured infectious viral titer (using a microfocus-forming assay) and total and subgenomic viral RNA levels (using RT-PCR) in a set of 162 clinical samples containing SARS-CoV-2 Alpha, Delta, and Epsilon variants that were collected in identical swab kits from outpatient test sites and processed soon after collection. We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite this, the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (5.9- and 3.0-fold increase; P < 0.0001, P = 0.014, respectively) or subgenomic E RNA (14.3- and 6.9-fold increase; P < 0.0001, P = 0.004, respectively). In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity for Delta may further explain increased spread, suggesting a need for increased measures to prevent viral transmission.


Subject(s)
COVID-19/epidemiology , Gene Expression Regulation, Viral , Genome, Viral , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Animals , COVID-19/pathology , COVID-19/transmission , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Hepatocytes/metabolism , Hepatocytes/virology , Humans , RNA, Viral/metabolism , SARS-CoV-2/classification , SARS-CoV-2/metabolism , Vero Cells , Viral Load , Virulence
3.
Infect Genet Evol ; 97: 105195, 2022 01.
Article in English | MEDLINE | ID: covidwho-1586990

ABSTRACT

SARS-CoV-2 is the RNA virus responsible for COVID-19, the prognosis of which has been found to be slightly worse in men. The present study aimed to analyze the expression of different mRNAs and their regulatory molecules (miRNAs and lncRNAs) to consider the potential existence of sex-specific expression patterns and COVID-19 susceptibility using bioinformatics analysis. The binding sites of all human mature miRNA sequences on the SARS-CoV-2 genome nucleotide sequence were predicted by the miRanda tool. Sequencing data was excavated using the Galaxy web server from GSE157103, and the output of feature counts was analyzed using DEseq2 packages to obtain differentially expressed genes (DEGs). Gene set enrichment analysis (GSEA) and DEG annotation analyses were performed using the ToppGene and Metascape tools. Using the RNA Interactome Database, we predicted interactions between differentially expressed lncRNAs and differentially expressed mRNAs. Finally, their networks were constructed with top miRNAs. We identified 11 miRNAs with three to five binding sites on the SARS-COVID-2 genome reference. MiR-29c-3p, miR-21-3p, and miR-6838-5p occupied four binding sites, and miR-29a-3p had five binding sites on the SARS-CoV-2 genome. Moreover, miR-29a-3p, and miR-29c-3p were the top miRNAs targeting DEGs. The expression levels of miRNAs (125, 181b, 130a, 29a, b, c, 212, 181a, 133a) changed in males with COVID-19, in whom they regulated ACE2 expression and affected the immune response by affecting phagosomes, complement activation, and cell-matrix adhesion. Our results indicated that XIST lncRNA was up-regulated, and TTTY14, TTTY10, and ZFY-AS1 lncRN as were down-regulated in both ICU and non-ICU men with COVID-19. Dysregulation of noncoding-RNAs has critical effects on the pathophysiology of men with COVID-19, which is why they may be used as biomarkers and therapeutic agents. Overall, our results indicated that the miR-29 family target regulation patterns and might become promising biomarkers for severity and survival outcome in men with COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , MicroRNAs/genetics , RNA, Long Noncoding/genetics , SARS-CoV-2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/epidemiology , COVID-19/pathology , COVID-19/virology , Computational Biology/methods , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Coronavirus M Proteins/genetics , Coronavirus M Proteins/metabolism , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Databases, Genetic , Female , Gene Expression Regulation , Host-Pathogen Interactions/genetics , Humans , Male , MicroRNAs/classification , MicroRNAs/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , RNA, Long Noncoding/classification , RNA, Long Noncoding/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , SARS-CoV-2/classification , SARS-CoV-2/pathogenicity , Severity of Illness Index , Sex Factors , Signal Transduction , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
4.
J Phys Chem Lett ; 12(51): 12249-12255, 2021 Dec 30.
Article in English | MEDLINE | ID: covidwho-1586057

ABSTRACT

SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratory's refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics that inhibit coronavirus budding.


Subject(s)
Coronavirus Envelope Proteins/metabolism , Molecular Dynamics Simulation , SARS-CoV-2/physiology , Viral Matrix Proteins/metabolism , COVID-19/pathology , COVID-19/virology , Coronavirus Envelope Proteins/chemistry , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Humans , Protein Multimerization , Protein Transport , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/chemistry
5.
Sci Rep ; 11(1): 24432, 2021 12 24.
Article in English | MEDLINE | ID: covidwho-1585772

ABSTRACT

Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1ß and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1ß, levels of IL-1ß and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy.


Subject(s)
Coronavirus Envelope Proteins/metabolism , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , SARS-CoV-2/metabolism , Animals , Bronchoalveolar Lavage Fluid/chemistry , COVID-19/pathology , COVID-19/virology , Coronavirus Envelope Proteins/genetics , Down-Regulation/drug effects , Endoplasmic Reticulum Stress , Humans , Inflammasomes/drug effects , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Janus Kinases/genetics , Janus Kinases/metabolism , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/deficiency , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Poly I-C/pharmacology , RNA, Viral/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/isolation & purification
6.
Science ; 374(6575): 1626-1632, 2021 Dec 24.
Article in English | MEDLINE | ID: covidwho-1501519

ABSTRACT

Efforts to determine why new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants demonstrate improved fitness have been limited to analyzing mutations in the spike (S) protein with the use of S-pseudotyped particles. In this study, we show that SARS-CoV-2 virus-like particles (SC2-VLPs) can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and at multiple steps in the viral life cycle. In SC2-VLPs, four nucleocapsid (N) mutations found universally in more-transmissible variants independently increased messenger RNA delivery and expression ~10-fold, and in a reverse genetics model, the serine-202→arginine (S202R) and arginine-203→methionine (R203M) mutations each produced >50 times as much virus. SC2-VLPs provide a platform for rapid testing of viral variants outside of a biosafety level 3 setting and demonstrate N mutations and particle assembly to be mechanisms that could explain the increased spread of variants, including B.1.617.2 (Delta, which contains the R203M mutation).


Subject(s)
Coronavirus Nucleocapsid Proteins/genetics , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Animals , Cell Line , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , Evolution, Molecular , Genome, Viral , Humans , Phosphoproteins/genetics , Phosphoproteins/metabolism , Plasmids , RNA, Messenger/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Genome Packaging , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Virus Internalization
7.
Mol Syst Biol ; 17(9): e10079, 2021 09.
Article in English | MEDLINE | ID: covidwho-1406892

ABSTRACT

We modeled 3D structures of all SARS-CoV-2 proteins, generating 2,060 models that span 69% of the viral proteome and provide details not available elsewhere. We found that ˜6% of the proteome mimicked human proteins, while ˜7% was implicated in hijacking mechanisms that reverse post-translational modifications, block host translation, and disable host defenses; a further ˜29% self-assembled into heteromeric states that provided insight into how the viral replication and translation complex forms. To make these 3D models more accessible, we devised a structural coverage map, a novel visualization method to show what is-and is not-known about the 3D structure of the viral proteome. We integrated the coverage map into an accompanying online resource (https://aquaria.ws/covid) that can be used to find and explore models corresponding to the 79 structural states identified in this work. The resulting Aquaria-COVID resource helps scientists use emerging structural data to understand the mechanisms underlying coronavirus infection and draws attention to the 31% of the viral proteome that remains structurally unknown or dark.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Host-Pathogen Interactions/genetics , Protein Processing, Post-Translational , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Transport Systems, Neutral/chemistry , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Binding Sites , COVID-19/genetics , COVID-19/metabolism , COVID-19/virology , Computational Biology/methods , Coronavirus Envelope Proteins/chemistry , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Models, Molecular , Molecular Mimicry , Neuropilin-1/chemistry , Neuropilin-1/genetics , Neuropilin-1/metabolism , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Interaction Mapping/methods , Protein Multimerization , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Viroporin Proteins/chemistry , Viroporin Proteins/genetics , Viroporin Proteins/metabolism , Virus Replication
9.
Future Microbiol ; 16: 107-118, 2021 01.
Article in English | MEDLINE | ID: covidwho-1389067

ABSTRACT

Viruses have caused the death of millions of people worldwide. Specifically, human viruses are grouped into 21 families, including the family of coronaviruses (CoVs). In December 2019, in Wuhan, China, a new human CoV was identified, SARS-CoV-2. The first step of the infection mechanism of the SARS-CoV-2 in the human host is adhesion, which occurs through the S glycoprotein that is found in diverse human organs. Another way through which SARS-CoV-2 could possibly attach to the host's cells is by means of the histo-blood group antigens. In this work, we have reviewed the mechanisms by which some viruses bind to the histo-blood group antigens, which could be related to the susceptibility of the individual and are dependent on the histo-blood group.


Subject(s)
Blood Group Antigens/metabolism , COVID-19/pathology , Spike Glycoprotein, Coronavirus/metabolism , Virus Attachment , Animals , Chiroptera/virology , Coronavirus Envelope Proteins/metabolism , Disease Susceptibility/blood , Genome, Viral/genetics , Glycoproteins/metabolism , Humans , SARS-CoV-2/genetics
10.
Cell Res ; 31(8): 847-860, 2021 08.
Article in English | MEDLINE | ID: covidwho-1387284

ABSTRACT

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.


Subject(s)
Antiviral Agents/metabolism , COVID-19/pathology , Coronavirus Envelope Proteins/metabolism , Respiratory Distress Syndrome/etiology , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Animals , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Apoptosis , COVID-19/complications , COVID-19/drug therapy , COVID-19/virology , Coronavirus Envelope Proteins/antagonists & inhibitors , Coronavirus Envelope Proteins/genetics , Cytokines/metabolism , Disease Models, Animal , Half-Life , Humans , Lung/metabolism , Lung/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutagenesis, Site-Directed , SARS-CoV-2/isolation & purification , SARS-CoV-2/pathogenicity , Spleen/metabolism , Spleen/pathology , Viral Load , Virulence
11.
Int J Mol Sci ; 22(16)2021 Aug 23.
Article in English | MEDLINE | ID: covidwho-1367852

ABSTRACT

The SARS-CoV-2 pseudovirus is a commonly used strategy that mimics certain biological functions of the authentic virus by relying on biological legitimacy at the molecular level. Despite the fact that spike (S), envelope (E), and membrane (M) proteins together wrap up the SARS-CoV-2 virion, most of the reported pseudotype viruses consist of only the S protein. Here, we report that the presence of E and M increased the virion infectivity by promoting the S protein priming. The S, E, and M (SEM)-coated pseudovirion is spherical, containing crown-like spikes on the surface. Both S and SEM pseudoviruses packaged the same amounts of viral RNA, but the SEM virus bound more efficiently to cells stably expressing the viral receptor human angiotensin-converting enzyme II (hACE2) and became more infectious. Using this SEM pseudovirus, we examined the infectivity and antigenic properties of the natural SARS-CoV-2 variants. We showed that some variants have higher infectivity than the original virus and that some render the neutralizing plasma with lower potency. These studies thus revealed possible mechanisms of the dissemination advantage of these variants. Hence, the SEM pseudovirion provides a useful tool to evaluate the viral infectivity and capability of convalescent sera in neutralizing specific SARS-CoV-2 S dominant variants.


Subject(s)
Antibodies, Viral/metabolism , COVID-19/immunology , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/pathogenicity , Viral Matrix Proteins/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/virology , Cell Line , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/immunology , Coronavirus Envelope Proteins/ultrastructure , Cricetinae , Humans , Microscopy, Electron, Transmission , Mutation , Neutralization Tests , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology , Viral Matrix Proteins/ultrastructure , Virion/genetics , Virion/immunology , Virion/metabolism , Virion/ultrastructure
12.
Biomolecules ; 11(8)2021 07 30.
Article in English | MEDLINE | ID: covidwho-1334993

ABSTRACT

The recent pandemic we are experiencing caused by the coronavirus disease 2019 (COVID-19) has put the world's population on the rack, with more than 191 million cases and more than 4.1 million deaths confirmed to date. This disease is caused by a new type of coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A massive proteomic analysis has revealed that one of the structural proteins of the virus, the E protein, interacts with BRD2 and BRD4 proteins of the Bromodomain and Extra Terminal domain (BET) family of proteins. BETs are essential to cell cycle progression, inflammation and immune response and have also been strongly associated with infection by different types of viruses. The fundamental role BET proteins play in transcription makes them appropriate targets for the propagation strategies of some viruses. Recognition of histone acetylation by BET bromodomains is essential for transcription control. The development of drugs mimicking acetyl groups, and thereby able to displace BET proteins from chromatin, has boosted interest on BETs as attractive targets for therapeutic intervention. The success of these drugs against a variety of diseases in cellular and animal models has been recently enlarged with promising results from SARS-CoV-2 infection studies.


Subject(s)
COVID-19/metabolism , Cell Cycle Proteins/metabolism , Transcription Factors/metabolism , COVID-19/immunology , COVID-19/virology , Coronavirus Envelope Proteins/metabolism , Humans , Immunity, Innate , Protein Binding
13.
J Biol Chem ; 297(2): 100940, 2021 08.
Article in English | MEDLINE | ID: covidwho-1293905

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 envelope protein (S2-E) is a conserved membrane protein that is important for coronavirus (CoV) assembly and budding. Here, we describe the recombinant expression and purification of S2-E in amphipol-class amphipathic polymer solutions, which solubilize and stabilize membrane proteins, but do not disrupt membranes. We found that amphipol delivery of S2-E to preformed planar bilayers results in spontaneous membrane integration and formation of viroporin cation channels. Amphipol delivery of the S2-E protein to human cells results in plasma membrane integration, followed by retrograde trafficking to the trans-Golgi network and accumulation in swollen perinuclear lysosomal-associated membrane protein 1-positive vesicles, likely lysosomes. CoV envelope proteins have previously been proposed to manipulate the luminal pH of the trans-Golgi network, which serves as an accumulation station for progeny CoV particles prior to cellular egress via lysosomes. Delivery of S2-E to cells will enable chemical biological approaches for future studies of severe acute respiratory syndrome coronavirus 2 pathogenesis and possibly even development of "Trojan horse" antiviral therapies. Finally, this work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.


Subject(s)
Cell Membrane/drug effects , Coronavirus Envelope Proteins/metabolism , Polymers/pharmacology , Propylamines/pharmacology , Surface-Active Agents/pharmacology , trans-Golgi Network/metabolism , Cell Membrane/metabolism , Coronavirus Envelope Proteins/genetics , HeLa Cells , Humans , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Lysosomes/metabolism , Polymers/chemistry , Propylamines/chemistry , Protein Transport , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Surface-Active Agents/chemistry
14.
Commun Biol ; 4(1): 724, 2021 06 11.
Article in English | MEDLINE | ID: covidwho-1265978

ABSTRACT

SARS-CoV-2 infection leads to coronavirus disease 2019 (COVID-19), which is associated with severe and life-threatening pneumonia and respiratory failure. However, the molecular basis of these symptoms remains unclear. SARS-CoV-1 E protein interferes with control of cell polarity and cell-cell junction integrity in human epithelial cells by binding to the PALS1 PDZ domain, a key component of the Crumbs polarity complex. We show that C-terminal PDZ binding motifs of SARS-CoV-1 and SARS-CoV-2 E proteins bind the PALS1 PDZ domain with 29.6 and 22.8 µM affinity, whereas the related sequence from MERS-CoV did not bind. We then determined crystal structures of PALS1 PDZ domain bound to both SARS-CoV-1 and SARS-CoV-2 E protein PDZ binding motifs. Our findings establish the structural basis for SARS-CoV-1/2 mediated subversion of Crumbs polarity signalling and serve as a platform for the development of small molecule inhibitors to suppress SARS-CoV-1/2 mediated disruption of polarity signalling in epithelial cells.


Subject(s)
Coronavirus Envelope Proteins/chemistry , Coronavirus Envelope Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Nucleoside-Phosphate Kinase/chemistry , Nucleoside-Phosphate Kinase/metabolism , PDZ Domains , Amino Acid Sequence , Humans , Models, Molecular , Protein Binding
15.
Cell Res ; 31(8): 847-860, 2021 08.
Article in English | MEDLINE | ID: covidwho-1265947

ABSTRACT

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.


Subject(s)
Antiviral Agents/metabolism , COVID-19/pathology , Coronavirus Envelope Proteins/metabolism , Respiratory Distress Syndrome/etiology , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Animals , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Apoptosis , COVID-19/complications , COVID-19/drug therapy , COVID-19/virology , Coronavirus Envelope Proteins/antagonists & inhibitors , Coronavirus Envelope Proteins/genetics , Cytokines/metabolism , Disease Models, Animal , Half-Life , Humans , Lung/metabolism , Lung/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutagenesis, Site-Directed , SARS-CoV-2/isolation & purification , SARS-CoV-2/pathogenicity , Spleen/metabolism , Spleen/pathology , Viral Load , Virulence
16.
PLoS One ; 16(6): e0251955, 2021.
Article in English | MEDLINE | ID: covidwho-1262543

ABSTRACT

Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway and/or gastrointestinal barrier damage and mitigate virus spread.


Subject(s)
COVID-19/metabolism , COVID-19/virology , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/metabolism , Zonula Occludens-1 Protein/metabolism , COVID-19/pathology , Host-Pathogen Interactions , Humans , PDZ Domains , Protein Binding , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SARS-CoV-2/isolation & purification , Tight Junctions/metabolism
17.
Nat Commun ; 12(1): 3433, 2021 06 08.
Article in English | MEDLINE | ID: covidwho-1261998

ABSTRACT

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has created global health and economic emergencies. SARS-CoV-2 viruses promote their own spread and virulence by hijacking human proteins, which occurs through viral protein recognition of human targets. To understand the structural basis for SARS-CoV-2 viral-host protein recognition, here we use cryo-electron microscopy (cryo-EM) to determine a complex structure of the human cell junction protein PALS1 and SARS-CoV-2 viral envelope (E) protein. Our reported structure shows that the E protein C-terminal DLLV motif recognizes a pocket formed exclusively by hydrophobic residues from the PDZ and SH3 domains of PALS1. Our structural analysis provides an explanation for the observation that the viral E protein recruits PALS1 from lung epithelial cell junctions. In addition, our structure provides novel targets for peptide- and small-molecule inhibitors that could block the PALS1-E interactions to reduce E-mediated virulence.


Subject(s)
Coronavirus Envelope Proteins/chemistry , Coronavirus Envelope Proteins/metabolism , Intercellular Junctions/metabolism , Membrane Proteins/metabolism , Nucleoside-Phosphate Kinase/metabolism , Amino Acid Sequence , Coronavirus Envelope Proteins/ultrastructure , Cryoelectron Microscopy , Humans , Protein Domains , SARS-CoV-2/physiology , Structural Homology, Protein , Structure-Activity Relationship
18.
PLoS Pathog ; 17(5): e1009519, 2021 05.
Article in English | MEDLINE | ID: covidwho-1232468

ABSTRACT

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.


Subject(s)
Amiloride/pharmacology , COVID-19/drug therapy , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Amiloride/pharmacokinetics , Animals , Antiviral Agents/pharmacology , Binding Sites/drug effects , COVID-19/virology , Chlorocebus aethiops , Coronavirus Envelope Proteins/chemistry , Humans , Ion Channels/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Binding/drug effects , Protein Conformation/drug effects , Protein Domains , Vero Cells , Virus Assembly/drug effects
19.
Nat Immunol ; 22(7): 829-838, 2021 07.
Article in English | MEDLINE | ID: covidwho-1220263

ABSTRACT

The innate immune response is critical for recognizing and controlling infections through the release of cytokines and chemokines. However, severe pathology during some infections, including SARS-CoV-2, is driven by hyperactive cytokine release, or a cytokine storm. The innate sensors that activate production of proinflammatory cytokines and chemokines during COVID-19 remain poorly characterized. In the present study, we show that both TLR2 and MYD88 expression were associated with COVID-19 disease severity. Mechanistically, TLR2 and Myd88 were required for ß-coronavirus-induced inflammatory responses, and TLR2-dependent signaling induced the production of proinflammatory cytokines during coronavirus infection independent of viral entry. TLR2 sensed the SARS-CoV-2 envelope protein as its ligand. In addition, blocking TLR2 signaling in vivo provided protection against the pathogenesis of SARS-CoV-2 infection. Overall, our study provides a critical understanding of the molecular mechanism of ß-coronavirus sensing and inflammatory cytokine production, which opens new avenues for therapeutic strategies to counteract the ongoing COVID-19 pandemic.


Subject(s)
COVID-19/immunology , Coronavirus Envelope Proteins/metabolism , Cytokine Release Syndrome/immunology , SARS-CoV-2/immunology , Toll-Like Receptor 2/metabolism , Animals , COVID-19/complications , COVID-19/diagnosis , COVID-19/drug therapy , COVID-19/virology , Chlorocebus aethiops , Cytokine Release Syndrome/diagnosis , Cytokines/metabolism , Disease Models, Animal , Female , Gene Expression Profiling , Humans , Immunity, Innate/drug effects , Leukocytes, Mononuclear , Macrophages , Male , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Primary Cell Culture , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Severity of Illness Index , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 2/antagonists & inhibitors , Toll-Like Receptor 2/genetics , Vero Cells
20.
Cells ; 10(4)2021 04 09.
Article in English | MEDLINE | ID: covidwho-1178118

ABSTRACT

Research on infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is currently restricted to BSL-3 laboratories. SARS-CoV2 virus-like particles (VLPs) offer a BSL-1, replication-incompetent system that can be used to evaluate virus assembly and virus-cell entry processes in tractable cell culture conditions. Here, we describe a SARS-CoV2 VLP system that utilizes nanoluciferase (Nluc) fragment complementation to track assembly and entry. We utilized the system in two ways. Firstly, we investigated the requirements for VLP assembly. VLPs were produced by concomitant synthesis of three viral membrane proteins, spike (S), envelope (E), and matrix (M), along with the cytoplasmic nucleocapsid (N). We discovered that VLP production and secretion were highly dependent on N proteins. N proteins from related betacoronaviruses variably substituted for the homologous SARS-CoV2 N, and chimeric betacoronavirus N proteins effectively supported VLP production if they contained SARS-CoV2 N carboxy-terminal domains (CTD). This established the CTDs as critical features of virus particle assembly. Secondly, we utilized the system by investigating virus-cell entry. VLPs were produced with Nluc peptide fragments appended to E, M, or N proteins, with each subsequently inoculated into target cells expressing complementary Nluc fragments. Complementation into functional Nluc was used to assess virus-cell entry. We discovered that each of the VLPs were effective at monitoring virus-cell entry, to various extents, in ways that depended on host cell susceptibility factors. Overall, we have developed and utilized a VLP system that has proven useful in identifying SARS-CoV2 assembly and entry features.


Subject(s)
COVID-19/metabolism , SARS-CoV-2/physiology , Virion/physiology , Virus Assembly , Virus Internalization , Coronavirus Envelope Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Nucleocapsid Proteins/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Viral Matrix Proteins/metabolism
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