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1.
Virology ; 568: 13-22, 2022 03.
Article in English | MEDLINE | ID: covidwho-1639193

ABSTRACT

Heightened inflammatory response is a prominent feature of severe COVID-19 disease. We report that the SARS-CoV-2 ORF3a viroporin activates the NLRP3 inflammasome, the most promiscuous of known inflammasomes. Ectopically expressed ORF3a triggers IL-1ß expression via NFκB, thus priming the inflammasome. ORF3a also activates the NLRP3 inflammasome but not NLRP1 or NLRC4, resulting in maturation of IL-1ß and cleavage/activation of Gasdermin. Notably, ORF3a activates the NLRP3 inflammasome via both ASC-dependent and -independent modes. This inflammasome activation requires efflux of potassium ions and oligomerization between the kinase NEK7 and NLRP3. Importantly, infection of epithelial cells with SARS-CoV-2 similarly activates the NLRP3 inflammasome. With the NLRP3 inhibitor MCC950 and select FDA-approved oral drugs able to block ORF3a-mediated inflammasome activation, as well as key ORF3a amino acid residues needed for virus release and inflammasome activation conserved in the new variants of SARS-CoV-2 isolates across continents, ORF3a and NLRP3 present prime targets for intervention.


Subject(s)
COVID-19/metabolism , COVID-19/virology , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , SARS-CoV-2/physiology , Signal Transduction , Viroporin Proteins/genetics , Amino Acid Sequence , Antiviral Agents/pharmacology , Cell Death , Cell Line , Host-Pathogen Interactions , Humans , Models, Biological , Open Reading Frames , Potassium/metabolism , Signal Transduction/drug effects , Viroporin Proteins/chemistry , Viroporin Proteins/metabolism
2.
Sci Rep ; 11(1): 13464, 2021 06 29.
Article in English | MEDLINE | ID: covidwho-1500743

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus disease 2019 (COVID-19) that emerged in human populations recently. Severely ill COVID-19 patients exhibit the elevation of proinflammatory cytokines, and such an unbalanced production of proinflammatory cytokines is linked to acute respiratory distress syndrome with high mortality in COVID-19 patients. Our study provides evidence that the ORF3a, M, ORF7a, and N proteins of SARS-CoV-2 were NF-κB activators. The viral sequence from infected zoo lions belonged to clade V, and a single mutation of G251V is found for ORF3a gene compared to all other clades. No significant functional difference was found for clade V ORF3a, indicating the NF-κB activation is conserved among COVID-19 variants. Of the four viral proteins, the ORF7a protein induced the NF-κB dictated proinflammatory cytokines including IL-1α, IL-1ß, IL-6, IL-8, IL-10, TNF-α, and IFNß. The ORF7a protein also induced IL-3, IL-4, IL-7, IL-23. Of 15 different chemokines examined in the study, CCL11, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL26, CCL27, and CXCL9 were significantly upregulated by ORF7. These cytokines and chemokines were frequently elevated in severely ill COVID-19 patients. Our data provide an insight into how SARS-CoV-2 modulates NF-κB signaling and inflammatory cytokine expressions. The ORF7a protein may be a desirable target for strategic developments to minimize uncontrolled inflammation in COVID-19 patients.


Subject(s)
Cytokines/metabolism , NF-kappa B/metabolism , SARS-CoV-2/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , COVID-19/pathology , COVID-19/virology , Chemokines/genetics , Chemokines/metabolism , Cytokines/genetics , HeLa Cells , Humans , Point Mutation , SARS-CoV-2/isolation & purification , Sequence Alignment , Severity of Illness Index , Up-Regulation , Viral Matrix Proteins/genetics , Viral Proteins/genetics , Viroporin Proteins/chemistry , Viroporin Proteins/genetics , Viroporin Proteins/metabolism
3.
Viruses ; 13(11)2021 10 27.
Article in English | MEDLINE | ID: covidwho-1488757

ABSTRACT

The current COVID-19 pandemic has highlighted the need for the research community to develop a better understanding of viruses, in particular their modes of infection and replicative lifecycles, to aid in the development of novel vaccines and much needed anti-viral therapeutics. Several viruses express proteins capable of forming pores in host cellular membranes, termed "Viroporins". They are a family of small hydrophobic proteins, with at least one amphipathic domain, which characteristically form oligomeric structures with central hydrophilic domains. Consequently, they can facilitate the transport of ions through the hydrophilic core. Viroporins localise to host membranes such as the endoplasmic reticulum and regulate ion homeostasis creating a favourable environment for viral infection. Viroporins also contribute to viral immune evasion via several mechanisms. Given that viroporins are often essential for virion assembly and egress, and as their structural features tend to be evolutionarily conserved, they are attractive targets for anti-viral therapeutics. This review discusses the current knowledge of several viroporins, namely Influenza A virus (IAV) M2, Human Immunodeficiency Virus (HIV)-1 Viral protein U (Vpu), Hepatitis C Virus (HCV) p7, Human Papillomavirus (HPV)-16 E5, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Open Reading Frame (ORF)3a and Polyomavirus agnoprotein. We highlight the intricate but broad immunomodulatory effects of these viroporins and discuss the current antiviral therapies that target them; continually highlighting the need for future investigations to focus on novel therapeutics in the treatment of existing and future emergent viruses.


Subject(s)
Immunomodulation , Ion Channels/metabolism , Viroporin Proteins/metabolism , Virus Diseases/drug therapy , Viruses/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Autophagy , Host-Pathogen Interactions , Human Immunodeficiency Virus Proteins/chemistry , Human Immunodeficiency Virus Proteins/metabolism , Immune Evasion , Inflammasomes/immunology , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/metabolism , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Viral Structural Proteins/chemistry , Viral Structural Proteins/metabolism , Viroporin Proteins/chemistry , Virus Diseases/immunology , Virus Diseases/virology , Viruses/drug effects , Viruses/immunology , Viruses/pathogenicity
4.
Proteins ; 89(12): 1987-1996, 2021 12.
Article in English | MEDLINE | ID: covidwho-1449944

ABSTRACT

Critical Assessment of Structure Prediction (CASP) is an organization aimed at advancing the state of the art in computing protein structure from sequence. In the spring of 2020, CASP launched a community project to compute the structures of the most structurally challenging proteins coded for in the SARS-CoV-2 genome. Forty-seven research groups submitted over 3000 three-dimensional models and 700 sets of accuracy estimates on 10 proteins. The resulting models were released to the public. CASP community members also worked together to provide estimates of local and global accuracy and identify structure-based domain boundaries for some proteins. Subsequently, two of these structures (ORF3a and ORF8) have been solved experimentally, allowing assessment of both model quality and the accuracy estimates. Models from the AlphaFold2 group were found to have good agreement with the experimental structures, with main chain GDT_TS accuracy scores ranging from 63 (a correct topology) to 87 (competitive with experiment).


Subject(s)
SARS-CoV-2/chemistry , Viral Proteins/chemistry , COVID-19/virology , Genome, Viral , Humans , Models, Molecular , Protein Conformation , Protein Domains , SARS-CoV-2/genetics , Viral Proteins/genetics , Viroporin Proteins/chemistry , Viroporin Proteins/genetics
5.
Sci Rep ; 11(1): 19481, 2021 09 30.
Article in English | MEDLINE | ID: covidwho-1447330

ABSTRACT

The pandemic infectious disease (Covid-19) caused by the coronavirus (SARS-CoV2) is spreading rapidly around the world. Covid-19 does an irreparable harm to the health and life of people. It also has a negative financial impact on the economies of most countries of the world. In this regard, the issue of creating drugs aimed at combating this disease is especially acute. In this work, molecular docking was used to study the docking of 23 compounds with QRF3a SARS-CoV2. The performed in silico modeling made it possible to identify leading compounds capable of exerting a potential inhibitory and virucidal effect. The leading compounds include chlorin (a drug used in PDT), iron(III)protoporphyrin (endogenous porphyrin), and tetraanthraquinone porphyrazine (an exogenous substance). Having taken into consideration the localization of ligands in the QRF3a SARS-CoV2, we have made an assumption about their influence on the pathogenesis of Covid-19. The interaction of chlorin, iron(III)protoporphyrin and protoporphyrin with the viral protein ORF3a were studied by fluorescence and UV-Vis spectroscopy. The obtained experimental results confirm the data of molecular docking. The results showed that a viral protein binds to endogenous porphyrins and chlorins, moreover, chlorin is a competitive ligand for endogenous porphyrins. Chlorin should be considered as a promising drug for repurposing.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/metabolism , Heterocyclic Compounds/chemistry , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/metabolism , Viroporin Proteins/chemistry , Viroporin Proteins/metabolism , Binding Sites , COVID-19/drug therapy , Drug Repositioning , Heterocyclic Compounds/metabolism , Ligands , Molecular Docking Simulation , Porphyrins/chemistry , Porphyrins/metabolism , Protoporphyrins/chemistry , Protoporphyrins/metabolism , SARS-CoV-2/drug effects , Viroporin Proteins/antagonists & inhibitors
6.
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
7.
Nat Struct Mol Biol ; 28(7): 573-582, 2021 07.
Article in English | MEDLINE | ID: covidwho-1279891

ABSTRACT

SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation and apoptosis. 3a protein and anti-3a antibodies are found in infected patient tissues and plasma. Deletion of 3a in SARS-CoV-1 reduces viral titer and morbidity in mice, suggesting it could be an effective target for vaccines or therapeutics. Here, we present structures of SARS-CoV-2 3a determined by cryo-EM to 2.1-Å resolution. 3a adopts a new fold with a polar cavity that opens to the cytosol and membrane through separate water- and lipid-filled openings. Hydrophilic grooves along outer helices could form ion-conduction paths. Using electrophysiology and fluorescent ion imaging of 3a-reconstituted liposomes, we observe Ca2+-permeable, nonselective cation channel activity, identify mutations that alter ion permeability and discover polycationic inhibitors of 3a activity. 3a-like proteins are found across coronavirus lineages that infect bats and humans, suggesting that 3a-targeted approaches could treat COVID-19 and other coronavirus diseases.


Subject(s)
Cryoelectron Microscopy , Nanostructures , SARS-CoV-2 , Viroporin Proteins/chemistry , Viroporin Proteins/ultrastructure , Animals , Calcium/metabolism , Chiroptera/virology , Coronaviridae , Electrophysiology , Fluorescence , Humans , Ion Transport , Liposomes , Models, Molecular , Nanostructures/chemistry , Nanostructures/ultrastructure , Open Reading Frames , Optical Imaging , Reproducibility of Results , SARS-CoV-2/chemistry , SARS-CoV-2/ultrastructure , Sequence Homology , Viral Proteins/chemistry , Viral Proteins/ultrastructure , Viroporin Proteins/antagonists & inhibitors
8.
Virus Res ; 300: 198441, 2021 07 15.
Article in English | MEDLINE | ID: covidwho-1221063

ABSTRACT

One of the most important proteins for COVID-19 pathogenesis in SARS-CoV-2 is the ORF3a which is the largest accessory protein among others coded by the SARS-CoV-2 genome. The major roles of the protein include virulence, infectivity, ion channel activity, morphogenesis, and virus release. The coronavirus, SARS-CoV-2 is mutating rapidly, therefore, critical study of mutations in ORF3a is certainly important from the pathogenic perspective. Here, a sum of 175 non-synonymous mutations in the ORF3a of SARS-CoV-2 were identified from 7194 complete genomes of SARS-CoV-2 available from NCBI database. Effects of these mutations on structural stability, and functions of ORF3a were also studied. Broadly, three different classes of mutations, such as neutral, disease, and mixed (neutral and disease) types of mutations were observed. Consecutive phenomena of mutations in ORF3a protein were studied based on the timeline of detection of the mutations. Considering the amino acid compositions of the ORF3a protein, twenty clusters were detected using the K-means clustering method. The present findings on 175 novel mutations of ORF3a proteins will extend our knowledge on ORF3a, a vital accessory protein in SARS-CoV-2, to enlighten the pathogenicity of this life-threatening virus.


Subject(s)
COVID-19/virology , SARS-CoV-2 , Viroporin Proteins , Virulence Factors , Databases, Genetic , Genes, Viral , Genetic Variation , Humans , Mutation, Missense , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Structure-Activity Relationship , Viroporin Proteins/chemistry , Viroporin Proteins/genetics , Virulence Factors/chemistry , Virulence Factors/genetics
9.
Biochim Biophys Acta Biomembr ; 1863(6): 183590, 2021 06 01.
Article in English | MEDLINE | ID: covidwho-1188312

ABSTRACT

The envelope protein E of the SARS-CoV coronavirus is an archetype of viroporin. It is a small hydrophobic protein displaying ion channel activity that has proven highly relevant in virus-host interaction and virulence. Ion transport through E channel was shown to alter Ca2+ homeostasis in the cell and trigger inflammation processes. Here, we study transport properties of the E viroporin in mixed solutions of potassium and calcium chloride that contain a fixed total concentration (mole fraction experiments). The channel is reconstituted in planar membranes of different lipid compositions, including a lipid mixture that mimics the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane where the virus localizes within the cell. We find that the E ion conductance changes non-monotonically with the total ionic concentration displaying an Anomalous Mole Fraction Effect (AMFE) only when charged lipids are present in the membrane. We also observe that E channel insertion in ERGIC-mimic membranes - including lipid with intrinsic negative curvature - enhances ion permeation at physiological concentrations of pure CaCl2 or KCl solutions, with a preferential transport of Ca2+ in mixed KCl-CaCl2 solutions. Altogether, our findings demonstrate that the presence of calcium modulates the transport properties of the E channel by interacting preferentially with charged lipids through different mechanisms including direct Coulombic interactions and possibly inducing changes in membrane morphology.


Subject(s)
Calcium/metabolism , SARS Virus/metabolism , Viroporin Proteins/metabolism , Amino Acid Sequence , Calcium Channels/metabolism , Ion Transport , Membrane Lipids/metabolism , Protein Binding , Protein Transport , Solutions , Viroporin Proteins/chemistry
10.
Protein Sci ; 30(6): 1114-1130, 2021 06.
Article in English | MEDLINE | ID: covidwho-1162948

ABSTRACT

The COVID-19 epidemic is one of the most influential epidemics in history. Understanding the impact of coronaviruses (CoVs) on host cells is very important for disease treatment. The SARS-CoV-2 envelope (E) protein is a small structural protein involved in many aspects of the viral life cycle. The E protein promotes the packaging and reproduction of the virus, and deletion of this protein weakens or even abolishes the virulence. This review aims to establish new knowledge by combining recent advances in the study of the SARS-CoV-2 E protein and by comparing it with the SARS-CoV E protein. The E protein amino acid sequence, structure, self-assembly characteristics, viroporin mechanisms and inhibitors are summarized and analyzed herein. Although the mechanisms of the SARS-CoV-2 and SARS-CoV E proteins are similar in many respects, specific studies on the SARS-CoV-2 E protein, for both monomers and oligomers, are still lacking. A comprehensive understanding of this protein should prompt further studies on the design and characterization of effective targeted therapeutic measures.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus Envelope Proteins/antagonists & inhibitors , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/physiology , Amino Acid Sequence , Animals , Antiviral Agents/chemistry , COVID-19/metabolism , COVID-19/virology , Coronavirus Envelope Proteins/chemistry , Humans , Models, Molecular , Protein Conformation , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , Sequence Alignment , Viroporin Proteins/antagonists & inhibitors , Viroporin Proteins/chemistry , Viroporin Proteins/metabolism
11.
Methods Enzymol ; 653: 207-235, 2021.
Article in English | MEDLINE | ID: covidwho-1051391

ABSTRACT

The SARS-CoV-2 3a protein is a putative ion channel implicated in virus life cycle and pathogenesis. We recently expressed, purified, and reconstituted 3a into lipid nanodiscs to solve its structure by cryo-EM to 2.1Å resolution. In this chapter, we describe methods we developed in order to facilitate the study of this protein in other laboratories. We emphasize factors that enabled rapid progression from gene sequence to reconstituted protein (3 weeks in the case of 3a) and provide general observations and tips for adapting these protocols to other membrane proteins of interest.


Subject(s)
Ion Channels/chemistry , Nanostructures , SARS-CoV-2/chemistry , Viral Envelope Proteins/chemistry , Viroporin Proteins/chemistry , Lipid Bilayers/chemistry
12.
PLoS One ; 16(1): e0238665, 2021.
Article in English | MEDLINE | ID: covidwho-1048815

ABSTRACT

This study aims to highlight SARS-COV-2 mutations which are associated with increased or decreased viral virulence. We utilize genetic data from all strains available from GISAID and countries' regional information, such as deaths and cases per million, as well as COVID-19-related public health austerity measure response times. Initial indications of selective advantage of specific mutations can be obtained from calculating their frequencies across viral strains. By applying modelling approaches, we provide additional information that is not evident from standard statistics or mutation frequencies alone. We therefore, propose a more precise way of selecting informative mutations. We highlight two interesting mutations found in genes N (P13L) and ORF3a (Q57H). The former appears to be significantly associated with decreased deaths and cases per million according to our models, while the latter shows an opposing association with decreased deaths and increased cases per million. Moreover, protein structure prediction tools show that the mutations infer conformational changes to the protein that significantly alter its structure when compared to the reference protein.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Viroporin Proteins/genetics , COVID-19/transmission , Coronavirus Nucleocapsid Proteins/chemistry , Geographic Information Systems , Humans , Linear Models , Mutation , Pandemics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phylogeny , Polymorphism, Single Nucleotide , SARS-CoV-2/classification , Viroporin Proteins/chemistry
13.
Int J Biol Macromol ; 170: 820-826, 2021 Feb 15.
Article in English | MEDLINE | ID: covidwho-996949

ABSTRACT

In this study, analysis of changes of SARS-CoV-2 ORF3a protein during pandemic is reported. ORF3a, a conserved coronavirus protein, is involved in virus replication and release. A set of 70,752 high-quality SARS-CoV-2 genomes available in GISAID databank at the end of August 2020 have been scanned. All ORF3a mutations in the virus genomes were grouped according to the collection date interval and over the entire data set. The considered intervals were: start of collection-February, March, April, May, June, July and August 2020. The top five most frequent variants were examined within each collection interval. Overall, seventeen variants have been isolated. Ten of the seventeen mutant sites occur within the transmembrane (TM) domain of ORF3a and are in contact with the central pore or side tunnels. The other variant sites are in different places of the ORF3a structure. Within the entire sample, the five most frequent mutations are V13L, Q57H, Q57H + A99V, G196V and G252V. The same analysis identified 28 sites identically conserved in all the genome isolates. These sites are possibly involved in stabilization of monomer, dimer, tetramerization and interaction with other cellular components. The results here reported can be helpful to understand virus biology and to design new therapeutic strategies.


Subject(s)
COVID-19/virology , Mutation , SARS-CoV-2/genetics , Viroporin Proteins/genetics , Amino Acid Sequence , COVID-19/epidemiology , Conserved Sequence , Databases, Genetic , Evolution, Molecular , Genome, Viral , Humans , Models, Molecular , Pandemics , Protein Structure, Quaternary , Proteome/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/physiology , Time Factors , Viroporin Proteins/chemistry , Viroporin Proteins/physiology
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