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1.
Biomolecules ; 14(6)2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38927063

RESUMO

The Ebola virus (EBOV) is a lethal pathogen causing hemorrhagic fever syndrome which remains a global health challenge. In the EBOV, two multifunctional proteins, VP35 and VP40, have significant roles in replication, virion assembly, and budding from the cell and have been identified as druggable targets. In this study, we employed in silico methods comprising molecular docking, molecular dynamic simulations, and pharmacological properties to identify prospective drugs for inhibiting VP35 and VP40 proteins from the myxobacterial bioactive natural product repertoire. Cystobactamid 934-2, Cystobactamid 919-1, and Cittilin A bound firmly to VP35. Meanwhile, 2-Hydroxysorangiadenosine, Enhypyrazinone B, and Sorangiadenosine showed strong binding to the matrix protein VP40. Molecular dynamic simulations revealed that, among these compounds, Cystobactamid 919-1 and 2-Hydroxysorangiadenosine had stable interactions with their respective targets. Similarly, molecular mechanics Poisson-Boltzmann surface area (MMPBSA) calculations indicated close-fitting receptor binding with VP35 or VP40. These two compounds also exhibited good pharmacological properties. In conclusion, we identified Cystobactamid 919-1 and 2-Hydroxysorangiadenosine as potential ligands for EBOV that target VP35 and VP40 proteins. These findings signify an essential step in vitro and in vivo to validate their potential for EBOV inhibition.


Assuntos
Antivirais , Produtos Biológicos , Ebolavirus , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ebolavirus/efeitos dos fármacos , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Antivirais/farmacologia , Antivirais/química , Myxococcales/química , Humanos , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/química , Proteínas do Nucleocapsídeo
2.
ACS Nano ; 18(24): 15545-15556, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38838261

RESUMO

Deterministic formation of membrane scission necks by protein machinery with multiplexed functions is critical in biology. A microbial example is M2 viroporin, a proton pump from the influenza A virus that is multiplexed with membrane remodeling activity to induce budding and scission in the host membrane during viral maturation. In comparison, the dynamin family constitutes a class of eukaryotic proteins implicated in mitochondrial fission, as well as various budding and endocytosis pathways. In the case of Dnm1, the mitochondrial fission protein in yeast, the membrane remodeling activity is multiplexed with mechanoenzyme activity to create fission necks. It is not clear why these functions are combined in these scission processes, which occur in drastically different compositions and solution conditions. In general, direct experimental access to changing neck sizes induced by individual proteins or peptide fragments is challenging due to the nanoscale dimensions and influence of thermal fluctuations. Here, we use a mechanical model to estimate the size of scission necks by leveraging small-angle X-ray scattering structural data of protein-lipid systems under different conditions. The influence of interfacial tension, lipid composition, and membrane budding morphology on the size of the induced scission necks is systematically investigated using our data and molecular dynamic simulations. We find that the M2 budding protein from the influenza A virus has robust pH-dependent membrane activity that induces nanoscopic necks within the range of spontaneous hemifission for a broad range of lipid compositions. In contrast, the sizes of scission necks generated by mitochondrial fission proteins strongly depend on lipid composition, which suggests a role for mechanical constriction.


Assuntos
Membrana Celular , Membrana Celular/metabolismo , Membrana Celular/química , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/química , Dinaminas/metabolismo , Dinaminas/química , Vírus da Influenza A/metabolismo , Espalhamento a Baixo Ângulo , Proteínas Viroporinas
3.
Virology ; 596: 110115, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38805802

RESUMO

Human cytomegalovirus (HCMV) replication relies on a nucleocapsid coat of the 150 kDa, subfamily-specific tegument phosphoprotein (pp150) to regulate cytoplasmic virion maturation. While recent structural studies revealed pp150-capsid interactions, the role of specific amino-acids involved in these interactions have not been established experimentally. In this study, pp150 and the small capsid protein (SCP), one of pp150's binding partners found atop the major capsid protein (MCP), were subjected to mutational and structural analyses. Mutations to clusters of polar or hydrophobic residues along the pp150-SCP interface abolished viral replication, with no replication detected in mutant virus-infected cells. Notably, a single amino acid mutation (pp150 K255E) at the pp150-MCP interface significantly attenuated viral replication, unlike in pp150-deletion mutants where capsids degraded outside host nuclei. These functionally significant mutations targeting pp150-capsid interactions, particularly the pp150 K255E replication-attenuated mutant, can be explored to overcome the historical challenges of developing effective antivirals and vaccines against HCMV infection.


Assuntos
Proteínas do Capsídeo , Citomegalovirus , Fosfoproteínas , Replicação Viral , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Humanos , Citomegalovirus/genética , Citomegalovirus/fisiologia , Citomegalovirus/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/química , Ligação Proteica , Mutagênese , Mutação , Linhagem Celular , Modelos Moleculares
4.
Protein Sci ; 33(5): e4978, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38591637

RESUMO

The Ebola virus (EBOV) is a lipid-enveloped virus with a negative sense RNA genome that can cause severe and often fatal viral hemorrhagic fever. The assembly and budding of EBOV is regulated by the matrix protein, VP40, which is a peripheral protein that associates with anionic lipids at the inner leaflet of the plasma membrane. VP40 is sufficient to form virus-like particles (VLPs) from cells, which are nearly indistinguishable from authentic virions. Due to the restrictions of studying EBOV in BSL-4 facilities, VP40 has served as a surrogate in cellular studies to examine the EBOV assembly and budding process from the host cell plasma membrane. VP40 is a dimer where inhibition of dimer formation halts budding and formation of new VLPs as well as VP40 localization to the plasma membrane inner leaflet. To better understand VP40 dimer stability and critical amino acids to VP40 dimer formation, we integrated computational approaches with experimental validation. Site saturation/alanine scanning calculation, combined with molecular mechanics-based generalized Born with Poisson-Boltzmann surface area (MM-GB/PBSA) method and molecular dynamics simulations were used to predict the energetic contribution of amino acids to VP40 dimer stability and the hydrogen bonding network across the dimer interface. These studies revealed several previously unknown interactions and critical residues predicted to impact VP40 dimer formation. In vitro and cellular studies were then pursued for a subset of VP40 mutations demonstrating reduction in dimer formation (in vitro) or plasma membrane localization (in cells). Together, the computational and experimental approaches revealed critical residues for VP40 dimer stability in an alpha-helical interface (between residues 106-117) as well as in a loop region (between residues 52-61) below this alpha-helical region. This study sheds light on the structural origins of VP40 dimer formation and may inform the design of a small molecule that can disrupt VP40 dimer stability.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Humanos , Ebolavirus/genética , Ebolavirus/metabolismo , Doença pelo Vírus Ebola/metabolismo , Membrana Celular/metabolismo , Simulação de Dinâmica Molecular , Aminoácidos/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo
5.
J Phys Chem B ; 128(11): 2595-2606, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38477117

RESUMO

The HIV-1 assembly process begins with a newly synthesized Gag polyprotein being targeted to the inner leaflet of the plasma membrane of the infected cells to form immature viral particles. Gag-membrane interactions are mediated through the myristoylated (Myr) N-terminal matrix (MA) domain of Gag, which eventually multimerize on the membrane to form trimers and higher order oligomers. The study of the structure and dynamics of peripheral membrane proteins like MA has been challenging for both experimental and computational studies due to the complex transient dynamics of protein-membrane interactions. Although the roles of anionic phospholipids (PIP2, PS) and the Myr group in the membrane targeting and stable membrane binding of MA are now well-established, the cooperative interactions between the MA monomers and MA-membrane remain elusive in the context of viral assembly and release. Our present study focuses on the membrane binding dynamics of a higher order oligomeric structure of MA protein (a dimer of trimers), which has not been explored before. Employing time-lagged independent component analysis (tICA) to our microsecond-long trajectories, we investigate conformational changes of the matrix protein induced by membrane binding. Interestingly, the Myr switch of an MA monomer correlates with the conformational switch of adjacent monomers in the same trimer. Together, our findings suggest complex protein dynamics during the formation of the immature HIV-1 lattice; while MA trimerization facilitates Myr insertion, MA trimer-trimer interactions in the immature lattice can hinder the same.


Assuntos
HIV-1 , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , HIV-1/metabolismo , Montagem de Vírus , Membrana Celular/metabolismo , Ligação Proteica , Proteínas da Matriz Viral/química
6.
J Biol Chem ; 300(5): 107213, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38522519

RESUMO

Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work, we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane-binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine and phosphatidylinositol 4,5-bisphosphate in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles. In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together, our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.


Assuntos
Membrana Celular , Ebolavirus , Montagem de Vírus , Liberação de Vírus , Humanos , Substituição de Aminoácidos , Membrana Celular/metabolismo , Ebolavirus/metabolismo , Ebolavirus/genética , Células HEK293 , Doença pelo Vírus Ebola/metabolismo , Doença pelo Vírus Ebola/virologia , Mutação , Nucleoproteínas , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/metabolismo , Fosfatidilserinas/química , Ligação Proteica , Eletricidade Estática , Proteínas do Core Viral/metabolismo , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/química , Vírion/metabolismo , Vírion/genética
7.
Molecules ; 29(3)2024 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-38338365

RESUMO

The influenza BM2 transmembrane domain (BM2TM), an acid-activated proton channel, is an attractive antiviral target due to its essential roles during influenza virus replication, whereas no effective inhibitors have been reported for BM2. In this study, we draw inspiration from the properties of cyclodextrins (CDs) and hypothesize that CDs of appropriate sizes may possess the potential to act as inhibitors of the BM2TM proton channel. To explore this possibility, molecular dynamics simulations were employed to assess their inhibitory capabilities. Our findings reveal that CD4, CD5, and CD6 are capable of binding to the BM2TM proton channel, resulting in disrupted water networks and reduced hydrogen bond occupancy between H19 and the solvent within the BM2TM channel necessary for proton conduction. Notably, CD4 completely obstructs the BM2TM water channel. Based on these observations, we propose that CD4, CD5, and CD6 individually contribute to diminishing the proton transfer efficiency of the BM2 protein, and CD4 demonstrates promising potential as an inhibitor for the BM2 proton channel.


Assuntos
Ciclodextrinas , Influenza Humana , Humanos , Prótons , Ciclodextrinas/farmacologia , Ciclodextrinas/metabolismo , Vírus da Influenza B/química , Vírus da Influenza B/metabolismo , Simulação de Dinâmica Molecular , Proteínas da Matriz Viral/química
8.
Angew Chem Int Ed Engl ; 62(47): e202309069, 2023 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-37733579

RESUMO

Viroporins are small ion channels in membranes of enveloped viruses that play key roles during viral life cycles. To use viroporins as drug targets against viral infection requires in-depth mechanistic understanding and, with that, methods that enable investigations under in situ conditions. Here, we apply surface-enhanced infrared absorption (SEIRA) spectroscopy to Influenza A M2 reconstituted within a solid-supported membrane, to shed light on the mechanics of its viroporin function. M2 is a paradigm of pH-activated proton channels and controls the proton flux into the viral interior during viral infection. We use SEIRA to track the large-scale reorientation of M2's transmembrane α-helices in situ during pH-activated channel opening. We quantify this event as a helical tilt from 26° to 40° by correlating the experimental results with solid-state nuclear magnetic resonance-informed computational spectroscopy. This mechanical motion is impeded upon addition of the inhibitor rimantadine, giving a direct spectroscopic marker to test antiviral activity. The presented approach provides a spectroscopic tool to quantify large-scale structural changes and to track the function and inhibition of the growing number of viroporins from pathogenic viruses in future studies.


Assuntos
Influenza Humana , Humanos , Prótons , Proteínas da Matriz Viral/química , Proteínas Viroporinas , Espectroscopia de Ressonância Magnética
9.
Bioconjug Chem ; 34(8): 1447-1458, 2023 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-37458383

RESUMO

The influenza A virus causes substantial morbidity and mortality worldwide every year and poses a constant threat of an emergent pandemic. Seasonal influenza vaccination strategies fail to provide complete protection against infection due to antigenic drift and shift. A universal vaccine targeting a conserved influenza epitope could substantially improve current vaccination strategies. The ectodomain of the matrix 2 protein (M2e) of influenza is a highly conserved epitope between virus strains but is also poorly immunogenic. Administration of M2e and the immunostimulatory stimulator of interferon genes (STING) agonist 3'3'-cyclic guanosine-adenosine monophosphate (cGAMP) encapsulated in microparticles made of acetalated dextran (Ace-DEX) has previously been shown to be effective for increasing the immunogenicity of M2e, primarily through T-cell-mediated responses. Here, the immunogenicity of Ace-DEX MPs delivering M2e was further improved by conjugating the M2e peptide to the particle surface in an effort to affect B-cell responses more directly. Conjugated or encapsulated M2e co-administered with Ace-DEX MPs containing cGAMP were used to vaccinate mice, and it was shown that two or three vaccinations could fully protect against a lethal influenza challenge, while only the surface-conjugated antigen constructs could provide some protection against lethal challenge with only one vaccination. Additionally, the use of a reducible linker augmented the T-cell response to the antigen. These results show the utility of conjugating M2e to the surface of a particle carrier to increase its immunogenicity for use as the antigen in a universal influenza vaccine.


Assuntos
Vírus da Influenza A , Vacinas contra Influenza , Influenza Humana , Animais , Camundongos , Humanos , Influenza Humana/prevenção & controle , Dextranos/química , Epitopos , Camundongos Endogâmicos BALB C , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Anticorpos Antivirais
10.
J Phys Chem B ; 127(29): 6449-6461, 2023 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-37458567

RESUMO

The Ebola virus (EBOV) is a filamentous virus that acquires its lipid envelope from the plasma membrane of the host cell it infects. EBOV assembly and budding from the host cell plasma membrane are mediated by a peripheral protein, known as the matrix protein VP40. VP40 is a 326 amino acid protein with two domains that are loosely linked. The VP40 N-terminal domain (NTD) contains a hydrophobic α-helix, which mediates VP40 dimerization. The VP40 C-terminal domain has a cationic patch, which mediates interactions with anionic lipids and a hydrophobic region that mediates VP40 dimer-dimer interactions. The VP40 dimer is necessary for trafficking to the plasma membrane inner leaflet and interactions with anionic lipids to mediate the VP40 assembly and oligomerization. Despite significant structural information available on the VP40 dimer structure, little is known on how the VP40 dimer is stabilized and how residues outside the NTD hydrophobic portion of the α-helical dimer interface contribute to dimer stability. To better understand how VP40 dimer stability is maintained, we performed computational studies using per-residue energy decomposition and site saturation mutagenesis. These studies revealed a number of novel keystone residues for VP40 dimer stability just adjacent to the α-helical dimer interface as well as distant residues in the VP40 CTD that can stabilize the VP40 dimer form. Experimental studies with representative VP40 mutants in vitro and in cells were performed to test computational predictions that reveal residues that alter VP40 dimer stability. Taken together, these studies provide important biophysical insights into VP40 dimerization and may be useful in strategies to weaken or alter the VP40 dimer structure as a means of inhibiting the EBOV assembly.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Humanos , Doença pelo Vírus Ebola/metabolismo , Ebolavirus/genética , Ebolavirus/metabolismo , Dimerização , Mutagênese , Lipídeos/química , Proteínas da Matriz Viral/química
11.
Structure ; 31(9): 1038-1051.e7, 2023 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-37392738

RESUMO

The Ebola virus matrix protein VP40 mediates viral budding and negatively regulates viral RNA synthesis. The mechanisms by which these two functions are exerted and regulated are unknown. Using a high-resolution crystal structure of Sudan ebolavirus (SUDV) VP40, we show here that two cysteines in the flexible C-terminal arm of VP40 form a stabilizing disulfide bridge. Notably, the two cysteines are targets of posttranslational redox modifications and interact directly with the host`s thioredoxin system. Mutation of the cysteines impaired the budding function of VP40 and relaxed its inhibitory role for viral RNA synthesis. In line with these results, the growth of recombinant Ebola viruses carrying cysteine mutations was impaired and the released viral particles were elongated. Our results revealed the exact positions of the cysteines in the C-terminal arm of SUDV VP40. The cysteines and/or their redox status are critically involved in the differential regulation of viral budding and viral RNA synthesis.


Assuntos
Ebolavirus , Proteínas da Matriz Viral , Ebolavirus/genética , Ebolavirus/metabolismo , Mutação , Oxirredução , Sudão , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Montagem de Vírus , Humanos
12.
ChemMedChem ; 18(16): e202300182, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37377066

RESUMO

We compared the anti-influenza potencies of 57 adamantyl amines and analogs against influenza A virus with serine-31 M2 proton channel, usually termed as WT M2 channel, which is amantadine sensitive. We also tested a subset of these compounds against viruses with the amantadine-resistant L26F, V27A, A30T, G34E M2 mutant channels. Four compounds inhibited WT M2 virus in vitro with mid-nanomolar potency, with 27 compounds showing sub-micromolar to low micromolar potency. Several compounds inhibited L26F M2 virus in vitro with sub-micromolar to low micromolar potency, but only three compounds blocked L26F M2-mediated proton current as determined by electrophysiology (EP). One compound was found to be a triple blocker of WT, L26F, V27A M2 channels by EP assays, but did not inhibit V27A M2 virus in vitro, and one compound inhibited WT, L26F, V27A M2 in vitro without blocking V27A M2 channel. One compound blocked only L26F M2 channel by EP, but did not inhibit virus replication. The triple blocker compound is as long as rimantadine, but could bind and block V27A M2 channel due to its larger girth as revealed by molecular dynamics simulations, while MAS NMR informed on the interaction of the compound with M2(18-60) WT or L26F or V27A.


Assuntos
Influenza Humana , Simulação de Dinâmica Molecular , Humanos , Antivirais/química , Aminas/farmacologia , Prótons , Mutação , Influenza Humana/tratamento farmacológico , Amantadina/farmacologia , Amantadina/uso terapêutico , Proteínas da Matriz Viral/química , Farmacorresistência Viral
13.
J Biomol Struct Dyn ; 41(3): 1062-1071, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-34913847

RESUMO

Intraviral protein-protein interactions are crucial for replication, pathogenicity, and viral assembly. Among these, virus assembly is a critical step as it regulates the arrangements of viral structural proteins and helps in the encapsulation of genomic material. SARS-CoV-2 structural proteins play an essential role in the self-rearrangement, RNA encapsulation, and mature virus particle formation. In SARS-CoV, the membrane protein interacts with the envelope and spike protein in Endoplasmic Reticulum Golgi Intermediate Complex (ERGIC) to form an assembly in the lipid bilayer, followed by membrane-ribonucleoprotein (nucleocapsid) interaction. In this study, we tried to understand the interaction of membrane protein's interaction with envelope, spike, and nucleocapsid proteins using protein-protein docking. Further, simulation studies were performed up to 100 ns to examine the stability of protein-protein complexes of Membrane-Envelope, Membrane-Spike, and Membrane-Nucleocapsid proteins. Prime MM-GBSA showed high binding energy calculations for the simulated structures than the docked complex. The interactions identified in our study will be of great importance, as it provides valuable insight into the protein-protein complex, which could be the potential drug targets for future studies.Communicated by Ramaswamy H. Sarma.


Assuntos
COVID-19 , Proteínas do Nucleocapsídeo , Humanos , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , SARS-CoV-2/metabolismo , Proteínas do Envelope Viral/química , Proteínas da Matriz Viral/química , Proteínas de Membrana , Simulação de Acoplamento Molecular
14.
Biophys Chem ; 293: 106943, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36495688

RESUMO

Hepatitis B virus core antigen (HBc) with the insertion of four external domains of the influenza A M2 protein (HBc/4M2e) form virus-like particles whose structure was studied using a combination of molecular modeling and cryo-electron microscopy (cryo-EM). It was also shown that self-assembling of the particles occurs inside bacterial cells, but despite the big inner volume of the core shell particle, purified HBc/4M2e contain an insignificant amount of bacterial proteins. It was shown that a fragment of the M2e corresponding to 4M2e insertion is prone to formation of amyloid-like fibrils. However, as the part of the immunodominant loop, M2e insertion does not show a tendency to intermolecular interaction. A full-atomic HBc-4M2e model with the resolution of about 3 Å (3.13 Å for particles of Т = 4 symmetry, 3.7 Å for particles of Т = 3 symmetry) was obtained by molecular modeling methods based on cryo-EM data.


Assuntos
Antígenos do Núcleo do Vírus da Hepatite B , Proteínas da Matriz Viral , Microscopia Crioeletrônica , Antígenos do Núcleo do Vírus da Hepatite B/química , Vírus da Hepatite B/química , Modelos Moleculares , Proteínas da Matriz Viral/química
15.
Biophys J ; 122(1): 90-98, 2023 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-36403086

RESUMO

The M2 proton channel of influenza A is embedded into the viral envelope and allows acidification of the virion when the external pH is lowered. In contrast, no outward proton conductance is observed when the internal pH is lowered, although outward current is observed at positive voltage. Residues Trp41 and Asp44 are known to play a role in preventing pH-driven outward conductance, but the mechanism for this is unclear. We investigate this issue using classical molecular dynamics simulations with periodic proton hops. When all key His37 residues are neutral, inward proton movement is much more facile than outward movement if the His are allowed to shuttle the proton. The preference for inward movement increases further as the charge on the His37 increases. Analysis of the trajectories reveals three factors accounting for this asymmetry. First, in the outward direction, Asp44 traps the hydronium by strong electrostatic interactions. Secondly, Asp44 and Trp41 orient the hydronium with the protons pointing inward, hampering outward Grotthus hopping. As a result, the effective barrier is lower in the inward direction. Trp41 adds to the barrier by weakly H-bonding to potential H+ acceptors. Finally, for charged His, the H3O+ in the inner vestibule tends to get trapped at lipid-lined fenestrations of the cone-shaped channel. Simulations qualitatively reproduce the experimentally observed higher outward conductance of mutants. The ability of positive voltage, unlike proton gradient, to induce an outward current appears to arise from its ability to bias H3O+ and the waters around it toward more H-outward orientations.


Assuntos
Vírus da Influenza A , Prótons , Proteínas da Matriz Viral , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Proteínas da Matriz Viral/química , Vírus da Influenza A/classificação
16.
J Virol ; 96(18): e0071622, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-36098511

RESUMO

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/genética
17.
J Virol ; 96(15): e0078622, 2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-35861516

RESUMO

The M1 of influenza A virus (IAV) is important for the virus life cycle, especially for the assembly and budding of viruses, which is a multistep process that requires host factors. Identifying novel host proteins that interact with M1 and understanding their functions in IAV replication are of great interest in antiviral drug development. In this study, we identified 19 host proteins in DF1 cells suspected to interact with the M1 protein of an H5N6 virus through immunoprecipitation (IP)/mass spectrometry. Among them, PSMD12, a 26S proteasome regulatory subunit, was shown to interact with influenza M1, acting as a positive host factor in IAV replication in avian and human cells. The data showed that PSMD12 promoted K63-linked ubiquitination of M1 at the K102 site. H5N6 and PR8 with an M1-K102 site mutant displayed a significantly weaker replication ability than the wild-type viruses. Mechanistically, PSMD12 promoted M1-M2 virus-like particle (VLP) release, and an M1-K102 mutation disrupted the formation of supernatant M1-M2 VLPs. An H5N6 M1-K102 site mutation or knockdown PSMD12 disrupted the budding release of the virus in chicken embryo fibroblast (CEF) cells, which was confirmed by transmission electron microscopy. Further study confirmed that M1-K102 site mutation significantly affected the virulence of H5N6 and PR8 viruses in mice. In conclusion, we report the novel host factor PSMD12 which affects the replication of influenza virus by mediating K63-linked ubiquitination of M1 at K102. These findings provide novel insight into the interactions between IAV and host cells, while suggesting an important target for anti-influenza virus drug research. IMPORTANCE M1 is proposed to play multiple biologically important roles in the life cycle of IAV, which relies largely on host factors. This study is the first one to identify that PSMD12 interacts with M1, mediates K63-linked ubiquitination of M1 at the K102 site, and thus positively regulates influenza virus proliferation. PSMD12 promoted M1-M2 VLP egress, and an M1-K102 mutation affected the M1-M2 VLP formation. Furthermore, we demonstrate the importance of this site to the morphology and budding of influenza viruses by obtaining mutant viruses, and the M1 ubiquitination regulator PSMD12 has a similar function to the M1 K102 mutation in regulating virus release and virus morphology. Additionally, we confirm the reduced virulence of H5N6 and PR8 (H1N1) viruses carrying the M1-K102 site mutation in mice. These findings provide novel insights into IAV interactions with host cells and suggest a valid and highly conserved candidate target for antiviral drug development.


Assuntos
Interações Hospedeiro-Patógeno , Vírus da Influenza A , Complexo de Endopeptidases do Proteassoma , Ubiquitinação , Proteínas da Matriz Viral , Replicação Viral , Animais , Antivirais , Linhagem Celular , Embrião de Galinha , Fibroblastos , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Camundongos , Mutação , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Virulência/genética
18.
Biochim Biophys Acta Biomembr ; 1864(7): 183909, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35276226

RESUMO

The influenza M2 protein forms a drug-targeted tetrameric proton channel to mediate virus uncoating, and carries out membrane scission to enable virus release. While the proton channel function of M2 has been extensively studied, the mechanism by which M2 catalyzes membrane scission is still not well understood. Previous fluorescence and electron microscopy studies indicated that M2 tetramers concentrate at the neck of the budding virus in the host plasma membrane. However, molecular evidence for this clustering is scarce. Here, we use 19F solid-state NMR to investigate M2 clustering in phospholipid bilayers. By mixing equimolar amounts of 4F-Phe47 labeled M2 peptide and CF3-Phe47 labeled M2 peptide and measuring F-CF3 cross peaks in 2D 19F19F correlation spectra, we show that M2 tetramers form nanometer-scale clusters in lipid bilayers. This clustering is stronger in cholesterol-containing membranes and phosphatidylethanolamine (PE) membranes than in cholesterol-free phosphatidylcholine and phosphatidylglycerol membranes. The observed correlation peaks indicate that Phe47 sidechains from different tetramers are less than ~2 nm apart. 1H19F correlation peaks between lipid chain protons and fluorinated Phe47 indicate that Phe47 is more deeply inserted into the lipid bilayer in the presence of cholesterol than in its absence, suggesting that Phe47 preferentially interacts with cholesterol. Static 31P NMR spectra indicate that M2 induces negative Gaussian curvature in the PE membrane. These results suggest that M2 tetramers cluster at cholesterol- and PE-rich regions of cell membranes to cause membrane curvature, which in turn can facilitate membrane scission in the last step of virus budding and release.


Assuntos
Influenza Humana , Bicamadas Lipídicas , Colesterol/química , Análise por Conglomerados , Humanos , Bicamadas Lipídicas/química , Peptídeos , Prótons , Proteínas da Matriz Viral/química
19.
Antiviral Res ; 199: 105267, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35227759

RESUMO

The central role of Ebola virus (EBOV) VP40 in nascent virion assembly and budding from infected host cells makes it an important therapeutic target. The mechanism of dimerization, following oligomerization of VP40 leading to the production of virus-like particles (VLP) has never been investigated for the development of therapeutic candidates against Ebola disease. Molecular dynamics-based computational screening targeted VP40 dimer with 40,000,000 compounds selected 374 compounds. A novel in vitro screening assay selected two compounds, NUSU#1 and NUSU#2. Conventional VLP assays consistently showed that both compounds inhibited EBOV VP40-mediated VLP production. Intriguingly, NUSU#1 inhibited the VP40-mediated VLP production in other ebolavirus species and the Marburg virus, but did not inhibit Lassa virus Z-mediated VLP production. These results strongly suggested that the selected compounds are potential lead drug candidates against Filovirus disease via disruption of VP40-mediated particle production.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Marburgvirus , Ebolavirus/química , Humanos , Proteínas da Matriz Viral/química , Liberação de Vírus
20.
Proc Natl Acad Sci U S A ; 119(13): e2025607119, 2022 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-35320040

RESUMO

SignificanceAlthough the need for a universal influenza vaccine has long been recognized, only a handful of candidates have been identified so far, with even fewer advancing in the clinical pipeline. The 24-amino acid ectodomain of M2 protein (M2e) has been developed over the past two decades. However, M2e-based vaccine candidates have shortcomings, including the need for several administrations and the lack of sustained antibody titers over time. We report here a vaccine targeting strategy that has the potential to confer sustained and strong protection upon a single shot of a small amount of M2e antigen. The current COVID-19 pandemic has highlighted the importance of developing versatile, powerful platforms for the rapid deployment of vaccines against any incoming threat.


Assuntos
COVID-19 , Vírus da Influenza A , Vacinas contra Influenza , Influenza Humana , Proteínas da Matriz Viral , Proteínas Viroporinas , Animais , Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , COVID-19/prevenção & controle , Células Dendríticas/imunologia , Humanos , Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Pandemias/prevenção & controle , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/imunologia , Proteínas Viroporinas/imunologia
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