RESUMO
The discussion has revolved around the derivatives of amino acids and peptides containing carbocycles and their potential antiviral activity in vitro against influenza A, hepatitis C viruses, and coronavirus. Studies conducted on cell cultures reveal that aminoadamantane amino acid derivatives exhibit the capacity to hinder the replication of viruses containing viroporins. Furthermore, certain compounds demonstrate potent virucidal activity with respect to influenza A/H5N1 and hepatitis C virus particles. A conceptual framework for viroporin inhibitors has been introduced, incorporating carbocyclic motifs as membranotropic carriers in the structure, alongside a functional segment comprised of amino acids and peptides. These components correspond to the interaction with the inner surface of the channel's pore or another target protein.
Assuntos
Aminoácidos , Antivirais , Peptídeos , Antivirais/farmacologia , Antivirais/química , Antivirais/síntese química , Humanos , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/síntese química , Aminoácidos/química , Aminoácidos/farmacologia , Animais , Hepacivirus/efeitos dos fármacos , Proteínas Viroporinas/antagonistas & inibidores , Proteínas Viroporinas/metabolismo , Proteínas Viroporinas/química , Vírus da Influenza A/efeitos dos fármacosRESUMO
The HIV-1-encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with host proteins to support the virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. Here, we report on the Vpu oligomeric organization under membrane and aqueous conditions and provide insights into how the Vpu environment affects the oligomer formation. For these studies, we designed a maltose-binding protein (MBP)-Vpu chimera protein and produced it in E. coli in soluble form. We analyzed this protein using analytical size-exclusion chromatography (SEC), negative staining electron microscopy (nsEM), and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, we found that MBP-Vpu formed stable oligomers in solution, seemingly driven by Vpu transmembrane domain self-association. A coarse modeling of nsEM data as well as SEC and EPR data suggests that these oligomers most likely are pentamers, similar to what was reported regarding membrane-bound Vpu. We also noticed reduced MBP-Vpu oligomer stability upon reconstitution of the protein in ß-DDM detergent and mixtures of lyso-PC/PG or DHPC/DHPG. In these cases, we observed greater oligomer heterogeneity, with MBP-Vpu oligomeric order generally lower than in solution; however, larger oligomers were also present. Notably, we found that in lyso-PC/PG, above a certain protein concentration, MBP-Vpu assembles into extended structures, which had not been reported for Vpu. Therefore, we captured various Vpu oligomeric forms, which can shed light on Vpu quaternary organization. Our findings could be useful in understanding Vpu organization and function in cellular membranes and could provide information regarding the biophysical properties of single-pass transmembrane proteins.
Assuntos
HIV-1 , Proteínas do Vírus da Imunodeficiência Humana , Proteínas Virais Reguladoras e Acessórias , Proteínas Viroporinas , Membrana Celular/metabolismo , Escherichia coli , HIV-1/química , Canais Iônicos/química , Proteínas do Vírus da Imunodeficiência Humana/química , Proteínas Viroporinas/química , Proteínas Virais Reguladoras e Acessórias/químicaRESUMO
A de novo assembly algorithm is provided to propose the assembly of bitopic transmembrane domains (TMDs) of membrane proteins. The algorithm is probed using, in particular, viral channel forming proteins (VCPs) such as M2 of influenza A virus, E protein of severe acute respiratory syndrome corona virus (SARS-CoV), 6K of Chikungunya virus (CHIKV), SH of human respiratory syncytial virus (hRSV), and Vpu of human immunodeficiency virus type 2 (HIV-2). The generation of the structures is based on screening a 7-dimensional space. Assembly of the TMDs can be achieved either by simultaneously docking the individual TMDs or via a sequential docking. Scoring based on estimated binding energies (EBEs) of the oligomeric structures is obtained by the tilt to decipher the handedness of the bundles. The bundles match especially well for all-atom models of M2 referring to an experimentally reported tetrameric bundle. Docking of helical poly-peptides to experimental structures of M2 and E protein identifies improving EBEs for positively charged (K,R,H) and aromatic amino acids (F,Y,W). Data are improved when using polypeptides for which the coordinates of the amino acids are adapted to the Cα coordinates of the respective experimentally derived structures of the TMDs of the target proteins.
Assuntos
Simulação de Acoplamento Molecular , Peptídeos , Proteínas Viroporinas , Humanos , Avaliação Pré-Clínica de Medicamentos , Peptídeos/química , Estrutura Terciária de Proteína , Proteínas Viroporinas/química , Domínios ProteicosRESUMO
Influenza A virus (IAV) assembly at the plasma membrane is orchestrated by at least five viral components, including hemagglutinin (HA), neuraminidase (NA), matrix (M1), the ion channel M2, and viral ribonucleoprotein (vRNP) complexes, although particle formation is observed with expression of only HA and/or NA. While these five viral components are expressed efficiently in primary human monocyte-derived macrophages (MDMs) upon IAV infection, this cell type does not support efficient HA-M2 association and IAV particle assembly at the plasma membrane. Both defects are specific to MDMs and can be reversed upon disruption of F-actin. However, the relationship between the two defects is unclear. Here, we examined whether M2 contributes to particle assembly in MDMs and if so, which region of M2 determines the susceptibility to the MDM-specific and actin-dependent suppression. An analysis using correlative fluorescence and scanning electron microscopy showed that an M2-deficient virus failed to form budding structures at the cell surface even after F-actin was disrupted, indicating that M2 is essential for virus particle formation at the MDM surface. Notably, proximity ligation analysis revealed that a single amino acid substitution in a Glu-Glu-Tyr sequence (residues 74 to 76) in the M2 cytoplasmic tail allowed the HA-M2 association to occur efficiently even in MDMs with intact actin cytoskeleton. This phenotype did not correlate with known phenotypes of the M2 substitution mutants regarding M1 interaction or vRNP packaging in epithelial cells. Overall, our study identified M2 as a target of MDM-specific restriction of IAV assembly, which requires the Glu-Glu-Tyr sequence in the cytoplasmic tail. IMPORTANCE Human MDMs represent a cell type that is nonpermissive to particle formation of influenza A virus (IAV). We previously showed that close proximity association between viral HA and M2 proteins is blocked in MDMs. However, whether MDMs express a restriction factor against IAV assembly or whether they lack a dependency factor promoting assembly remained unknown. In the current study, we determined that the M2 protein is necessary for particle formation in MDMs but is also a molecular target of the MDM-specific suppression of assembly. Substitutions in the M2 cytoplasmic tail alleviated the block in both the HA-M2 association and particle production in MDMs. These findings suggest that MDMs express dependency factors necessary for assembly but also express a factor(s) that inhibits HA-M2 association and particle formation. High conservation of the M2 sequence rendering the susceptibility to the assembly block highlights the potential for M2 as a target of antiviral strategies.
Assuntos
Ácido Glutâmico , Hemaglutininas , Vírus da Influenza A , Macrófagos , Tirosina , Proteínas da Matriz Viral , Proteínas Viroporinas , Montagem de Vírus , Actinas/metabolismo , Sequência de Aminoácidos , Ácido Glutâmico/genética , Hemaglutininas/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Macrófagos/virologia , Neuraminidase/genética , Neuraminidase/metabolismo , Ribonucleoproteínas/genética , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Proteínas Viroporinas/química , Proteínas Viroporinas/metabolismo , Montagem de Vírus/genéticaRESUMO
Recent studies identified signal peptidase complex subunit 1 (SPCS1) as a proviral host factor for Flaviviridae viruses, including HCV. One of the SPCS1's roles in flavivirus propagation was attributed to its regulation of signal peptidase complex (SPC)-mediated processing of flavivirus polyprotein, especially C-prM junction. However, whether SPCS1 also regulates any SPC-mediated processing sites within HCV polyprotein remains unclear. In this study, we determined that loss of SPCS1 specifically impairs the HCV E2-p7 processing by the SPC. We also determined that efficient separation of E2 and p7, regardless of its dependence on SPC-mediated processing, leads to SPCS1 dispensable for HCV assembly These results suggest that SPCS1 regulates HCV assembly by facilitating the SPC-mediated processing of E2-p7 precursor. Structural modeling suggests that intrinsically delayed processing of the E2-p7 is likely caused by the structural rigidity of p7 N-terminal transmembrane helix-1 (p7/TM1/helix-1), which has mostly maintained membrane-embedded conformations during molecular dynamics (MD) simulations. E2-p7-processing-impairing p7 mutations narrowed the p7/TM1/helix-1 bending angle against the membrane, resulting in closer membrane embedment of the p7/TM1/helix-1 and less access of E2-p7 junction substrate to the catalytic site of the SPC, located well above the membrane in the ER lumen. Based on these results we propose that the key mechanism of action of SPCS1 in HCV assembly is to facilitate the E2-p7 processing by enhancing the E2-p7 junction site presentation to the SPC active site. By providing evidence that SPCS1 facilitates HCV assembly by regulating SPC-mediated cleavage of E2-p7 junction, equivalent to the previously established role of this protein in C-prM junction processing in flavivirus, this study establishes the common role of SPCS1 in Flaviviridae family virus propagation as to exquisitely regulate the SPC-mediated processing of specific, suboptimal target sites.
Assuntos
Hepacivirus/metabolismo , Hepatite C/virologia , Proteínas de Membrana/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Viroporinas/metabolismo , Montagem de Vírus , Linhagem Celular , Células HEK293 , Interações entre Hospedeiro e Microrganismos , Humanos , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Conformação Proteica , Proteínas do Envelope Viral/química , Proteínas Viroporinas/química , Replicação ViralRESUMO
The influenza A M2 channel, a prototype for viroporins, is an acid-activated viroporin that conducts protons across the viral membrane, a critical step in the viral life cycle. Four central His37 residues control channel activation by binding subsequent protons from the viral exterior, which opens the Trp41 gate and allows proton flux to the interior. Asp44 is essential for maintaining the Trp41 gate in a closed state at high pH, resulting in asymmetric conduction. The prevalent D44N mutant disrupts this gate and opens the C-terminal end of the channel, resulting in increased conduction and a loss of this asymmetric conduction. Here, we use extensive Multiscale Reactive Molecular Dynamics (MS-RMD) and quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations with an explicit, reactive excess proton to calculate the free energy of proton transport in this M2 mutant and to study the dynamic molecular-level behavior of D44N M2. We find that this mutation significantly lowers the barrier of His37 deprotonation in the activated state and shifts the barrier for entry to the Val27 tetrad. These free energy changes are reflected in structural shifts. Additionally, we show that the increased hydration around the His37 tetrad diminishes the effect of the His37 charge on the channel's water structure, facilitating proton transport and enabling activation from the viral interior. Altogether, this work provides key insight into the fundamental characteristics of PT in WT M2 and how the D44N mutation alters this PT mechanism, and it expands understanding of the role of emergent mutations in viroporins.
Assuntos
Vírus da Influenza A/metabolismo , Proteínas da Matriz Viral/metabolismo , Proteínas Viroporinas/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Prótons , Teoria Quântica , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas Viroporinas/química , Proteínas Viroporinas/genética , Água/químicaRESUMO
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.
Assuntos
COVID-19/metabolismo , COVID-19/virologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , SARS-CoV-2/fisiologia , Transdução de Sinais , Proteínas Viroporinas/genética , Sequência de Aminoácidos , Antivirais/farmacologia , Morte Celular , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Modelos Biológicos , Fases de Leitura Aberta , Potássio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Viroporinas/química , Proteínas Viroporinas/metabolismoRESUMO
The NLRP3 inflammasome consists of NLRP3, ASC, and pro-caspase-1 and is an important arm of the innate immune response against influenza A virus (IAV) infection. Upon infection, the inflammasome is activated, resulting in the production of IL-1ß and IL-18, which recruits other immune cells to the site of infection. It has been suggested that in the presence of stress molecules such as nigericin, the trans-Golgi network (TGN) disperses into small puncta-like structures where NLRP3 is recruited and activated. Here, we investigated whether IAV infection could lead to TGN dispersion, whether dispersed TGN (dTGN) is responsible for NLRP3 inflammasome activation, and which viral protein is involved in this process. We showed that the IAV causes dTGN formation, which serves as one of the mechanisms of NLRP3 inflammasome activation in response to IAV infection. Furthermore, we generated a series of mutant IAVs that carry mutations in the M2 protein. We demonstrated the M2 proton channel activity, specifically His37 and Trp41 are pivotal for the dispersion of TGN, NLRP3 conformational change, and IL-1ß induction. The results revealed a novel mechanism behind the activation and regulation of the NLRP3 inflammasome in IAV infection.
Assuntos
Inflamassomos/imunologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Rede trans-Golgi/fisiologia , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Células Cultivadas , Cães , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/imunologia , Interleucina-1beta/biossíntese , Mutação , Suínos , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Viroporinas/química , Proteínas Viroporinas/genética , Proteínas Viroporinas/metabolismo , Rede trans-Golgi/ultraestruturaRESUMO
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.
Assuntos
Imunomodulação , Canais Iônicos/metabolismo , Proteínas Viroporinas/metabolismo , Viroses/tratamento farmacológico , Vírus/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , Autofagia , Interações Hospedeiro-Patógeno , Proteínas do Vírus da Imunodeficiência Humana/química , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Evasão da Resposta Imune , Inflamassomos/imunologia , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/metabolismo , Proteínas Viroporinas/química , Viroses/imunologia , Viroses/virologia , Vírus/efeitos dos fármacos , Vírus/imunologia , Vírus/patogenicidadeRESUMO
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.
Assuntos
Antivirais/química , Antivirais/metabolismo , Compostos Heterocíclicos/química , Compostos Macrocíclicos/química , Compostos Macrocíclicos/metabolismo , Proteínas Viroporinas/química , Proteínas Viroporinas/metabolismo , Sítios de Ligação , Reposicionamento de Medicamentos , Compostos Heterocíclicos/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Porfirinas/química , Porfirinas/metabolismo , Protoporfirinas/química , Protoporfirinas/metabolismo , SARS-CoV-2/efeitos dos fármacos , Proteínas Viroporinas/antagonistas & inibidores , Tratamento Farmacológico da COVID-19RESUMO
Ion channels located at viral envelopes (viroporins) have a critical function for the replication of infectious viruses and are important drug targets. Over the last decade, the number and duration of molecular dynamics (MD) simulations of the influenza A M2 ion channel owing to the increased computational efficiency. Here, we aimed to define the system setup and simulation conditions for the correct description of the protein-pore and the protein-lipid interactions for influenza A M2 in comparison with experimental data. We performed numerous MD simulations of the influenza A M2 protein in complex with adamantane blockers in standard lipid bilayers using OPLS2005 and CHARMM36 (C36) force fields. We explored the effect of varying the M2 construct (M2(22-46) and M2(22-62)), the lipid buffer size and type (stiffer DMPC or softer POPC with or without 20% cholesterol), the simulation time, the H37 protonation site (Nδ or Νε), the conformational state of the W41 channel gate, and M2's cholesterol binding sites (BSs). We report that the 200 ns MD with M2(22-62) (having Nε Η37) in the 20 Å lipid buffer with the C36 force field accurately describe: (a) the M2 pore structure and interactions inside the pore, that is, adamantane channel blocker location, water clathrate structure, and water or chloride anion blockage/passage from the M2 pore in the presence of a channel blocker and (b) interactions between M2 and the membrane environment as reflected by the calculation of the M2 bundle tilt, folding of amphipathic helices, and cholesterol BSs. Strikingly, we also observed that the C36 1 µs MD simulations using M2(22-62) embedded in a 20 Å POPC:cholesterol (5:1) scrambled membrane produced frequent interactions with cholesterol, which when combined with computational kinetic analysis, revealed the experimentally observed BSs of cholesterol and suggested three similarly long-interacting positions in the top leaflet that have previously not been observed experimentally. These findings promise to be useful for other viroporin systems.
Assuntos
Vírus da Influenza A , Proteínas Viroporinas/química , Humanos , Influenza Humana , Cinética , Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Proteínas da Matriz ViralRESUMO
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 Ë6% of the proteome mimicked human proteins, while Ë7% was implicated in hijacking mechanisms that reverse post-translational modifications, block host translation, and disable host defenses; a further Ë29% 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.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Interações Hospedeiro-Patógeno/genética , Processamento de Proteína Pós-Traducional , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Sítios de Ligação , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Biologia Computacional/métodos , Proteínas do Envelope de Coronavírus/química , Proteínas do Envelope de Coronavírus/genética , Proteínas do Envelope de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Humanos , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Moleculares , Mimetismo Molecular , Neuropilina-1/química , Neuropilina-1/genética , Neuropilina-1/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Multimerização Proteica , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Viroporinas/química , Proteínas Viroporinas/genética , Proteínas Viroporinas/metabolismo , Replicação ViralRESUMO
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).
Assuntos
SARS-CoV-2/química , Proteínas Virais/química , COVID-19/virologia , Genoma Viral , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , SARS-CoV-2/genética , Proteínas Virais/genética , Proteínas Viroporinas/química , Proteínas Viroporinas/genéticaRESUMO
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.
Assuntos
Microscopia Crioeletrônica , Nanoestruturas , SARS-CoV-2 , Proteínas Viroporinas/química , Proteínas Viroporinas/ultraestrutura , Animais , Cálcio/metabolismo , Quirópteros/virologia , Coronaviridae , Eletrofisiologia , Fluorescência , Humanos , Transporte de Íons , Lipossomos , Modelos Moleculares , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Fases de Leitura Aberta , Imagem Óptica , Reprodutibilidade dos Testes , SARS-CoV-2/química , SARS-CoV-2/ultraestrutura , Homologia de Sequência , Proteínas Virais/química , Proteínas Virais/ultraestrutura , Proteínas Viroporinas/antagonistas & inibidoresRESUMO
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.
Assuntos
Canais Iônicos/química , Nanoestruturas , SARS-CoV-2/química , Proteínas do Envelope Viral/química , Proteínas Viroporinas/química , Bicamadas Lipídicas/químicaRESUMO
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.
Assuntos
Citocinas/metabolismo , NF-kappa B/metabolismo , SARS-CoV-2/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , COVID-19/patologia , COVID-19/virologia , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Células HeLa , Humanos , Mutação Puntual , SARS-CoV-2/isolamento & purificação , Alinhamento de Sequência , Índice de Gravidade de Doença , Regulação para Cima , Proteínas da Matriz Viral/genética , Proteínas Virais/genética , Proteínas Viroporinas/química , Proteínas Viroporinas/genética , Proteínas Viroporinas/metabolismoRESUMO
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.
Assuntos
COVID-19/virologia , SARS-CoV-2 , Proteínas Viroporinas , Fatores de Virulência , Bases de Dados Genéticas , Genes Virais , Variação Genética , Humanos , Mutação de Sentido Incorreto , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Relação Estrutura-Atividade , Proteínas Viroporinas/química , Proteínas Viroporinas/genética , Fatores de Virulência/química , Fatores de Virulência/genéticaRESUMO
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.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Proteínas do Envelope de Coronavírus/antagonistas & inibidores , Proteínas do Envelope de Coronavírus/metabolismo , SARS-CoV-2/fisiologia , Sequência de Aminoácidos , Animais , Antivirais/química , COVID-19/metabolismo , COVID-19/virologia , Proteínas do Envelope de Coronavírus/química , Humanos , Modelos Moleculares , Conformação Proteica , SARS-CoV-2/química , SARS-CoV-2/efeitos dos fármacos , Alinhamento de Sequência , Proteínas Viroporinas/antagonistas & inibidores , Proteínas Viroporinas/química , Proteínas Viroporinas/metabolismoRESUMO
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.
Assuntos
Cálcio/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Proteínas Viroporinas/metabolismo , Sequência de Aminoácidos , Canais de Cálcio/metabolismo , Transporte de Íons , Lipídeos de Membrana/metabolismo , Ligação Proteica , Transporte Proteico , Soluções , Proteínas Viroporinas/químicaRESUMO
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.