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1.
PLoS One ; 15(7): e0235483, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32697773

RESUMO

A series of potent HIV-1 protease inhibitors, containing diverse piperidine analogues as the P2-ligands, 4-substituted phenylsulfonamides as the P2'-ligands and a hydrophobic cyclopropyl group as the P1'-ligand, were designed, synthesized and evaluated in this work. Among these twenty-four target compounds, many of them exhibited excellent activity against HIV-1 protease with half maximal inhibitory concentration (IC50) values below 20 nM. Particularly, compound 22a containing a (R)-piperidine-3-carboxamide as the P2-ligand and a 4-methoxylphenylsulfonamide as the P2'-ligand exhibited the most effective inhibitory activity with an IC50 value of 3.61 nM. More importantly, 22a exhibited activity with inhibition of 42% and 26% against wild-type and Darunavir (DRV)-resistant HIV-1 variants, respectively. Additionally, the molecular docking of 22a with HIV-1 protease provided insight into the ligand-binding properties, which was of great value for further study.


Assuntos
Inibidores Enzimáticos/química , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/química , HIV-1/efeitos dos fármacos , Piperidinas/farmacologia , Cristalografia por Raios X , Darunavir/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Infecções por HIV/virologia , Protease de HIV/química , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/farmacologia , HIV-1/química , HIV-1/patogenicidade , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Sulfonamidas/farmacologia
2.
Proc Natl Acad Sci U S A ; 117(31): 18719-18728, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690692

RESUMO

CD4-based decoy approaches against HIV-1 are attractive options for long-term viral control, but initial designs, including soluble CD4 (sCD4) and CD4-Ig, were ineffective. To evaluate a therapeutic that more accurately mimics HIV-1 target cells compared with monomeric sCD4 and dimeric CD4-Ig, we generated virus-like nanoparticles that present clusters of membrane-associated CD4 (CD4-VLPs) to permit high-avidity binding of trimeric HIV-1 envelope spikes. In neutralization assays, CD4-VLPs were >12,000-fold more potent than sCD4 and CD4-Ig and >100-fold more potent than the broadly neutralizing antibody (bNAb) 3BNC117, with >12,000-fold improvements against strains poorly neutralized by 3BNC117. CD4-VLPs also neutralized patient-derived viral isolates that were resistant to 3BNC117 and other bNAbs. Intraperitoneal injections of CD4-CCR5-VLP produced only subneutralizing plasma concentrations in HIV-1-infected humanized mice but elicited CD4-binding site mutations that reduced viral fitness. All mutant viruses showed reduced sensitivity to sCD4 and CD4-Ig but remained sensitive to neutralization by CD4-VLPs in vitro. In vitro evolution studies demonstrated that CD4-VLPs effectively controlled HIV-1 replication at neutralizing concentrations, and viral escape was not observed. Moreover, CD4-VLPs potently neutralized viral swarms that were completely resistant to CD4-Ig, suggesting that escape pathways that confer resistance against conventional CD4-based inhibitors are ineffective against CD4-VLPs. These findings suggest that therapeutics that mimic HIV-1 target cells could prevent viral escape by exposing a universal vulnerability of HIV-1: the requirement to bind CD4 on a target cell. We propose that therapeutic and delivery strategies that ensure durable bioavailability need to be developed to translate this concept into a clinically feasible functional cure therapy.


Assuntos
Antígenos CD4 , HIV-1 , Nanopartículas , Vírion , Fármacos Anti-HIV , Antígenos CD4/química , Antígenos CD4/metabolismo , Linhagem Celular , HIV-1/química , HIV-1/genética , HIV-1/metabolismo , Humanos , Mimetismo Molecular , Nanomedicina/métodos , Nanopartículas/química , Nanopartículas/metabolismo , Vírion/química , Vírion/metabolismo
3.
Arch Biochem Biophys ; 688: 108401, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32376316

RESUMO

HIV-1 glycoprotein 41 (gp41) mediates fusion between virus and target cells by folding into a fusion active state, in which the C-terminal heptad repeat (CHR) regions associate externally to the N-terminal heptad repeat (NHR) trimer and form a very stable six-helix bundle coiled-coil structure. Therefore, interfering with the NHR-CHR interaction of gp41 is a promising therapeutic approach against HIV-1. However, a full understanding of the molecular and mechanistic details of this interaction is still incomplete. Here, we use single-chain, chimeric proteins (named covNHR) that reproduce accurately the CHR-NHR interactions to analyze the binding thermodynamics of several peptides with different length from the CHR region. The results indicate that cooperative binding involving two or more pockets of the NHR groove is necessary to obtain relevant affinities and that the binding energy is broadly distributed along the interface, underlining a crucial role of a middle pocket to achieve tight binding. In contrast, targeting only the deep hydrophobic pocket is insufficient to achieve significant affinity. Moreover, calorimetry experiments in combination with limited proteolysis performed using a mutant with occluded binding in the N-terminal pocket reveal the existence of an allosteric communication between the different regions. This study is the first detailed thermodynamic dissection of the NHR-CHR interaction in gp41 and contributes therefore to a better understanding of HIV fusion. These results are relevant for the development of potential fusion inhibitors.


Assuntos
Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/química , Fragmentos de Peptídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Calorimetria , Proteína gp41 do Envelope de HIV/química , Fragmentos de Peptídeos/química , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Termodinâmica
4.
Virology ; 546: 1-12, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32275203

RESUMO

Broadly neutralizing antibodies (bNAbs) may constitute an essential component of a protective vaccine against HIV-1, yet no immunogen has been able to elicit them. To characterize the development of bNAbs in HIV-1 subtype C infected individuals, a panel of 18 Env-pseudotyped viruses was used to screen 18 study participants. The specificity of plasma neutralization was mapped against Env mutants and MPER chimeras. Envelope (env) gene sequence evolution was characterized by single genome amplification and sequencing. Three out of eighteen individuals developed broad plasma neutralizing activity (>60% breadth). Two of the three participants may target epitopes comprising glycans at position 276 of the D loop in the CD4 binding site and 332 glycan supersite, respectively. Deletion of these glycans was associated with neutralization resistance. Our study describes the kinetics of the development of plasma neutralizing activity and identified amino acid residue changes suggestive of immune pressure on putative epitopes. The study enhances our understanding of how neutralization breadth develops in the course of HIV-1 subtype C infection.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Sequência de Aminoácidos , Mapeamento de Epitopos , Infecções por HIV/sangue , Infecções por HIV/virologia , HIV-1/química , HIV-1/classificação , HIV-1/genética , Humanos , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
5.
Nat Methods ; 17(3): 279-282, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066961

RESUMO

We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.


Assuntos
Proteínas de Fluorescência Verde/química , Fragmentos de Imunoglobulinas/química , Nanopartículas/química , Anticorpos de Domínio Único/química , Cristalografia por Raios X , DNA/química , Bases de Dados de Proteínas , Escherichia coli , Transferência Ressonante de Energia de Fluorescência , Produtos do Gene gag/química , Células HEK293 , HIV-1/química , Células HeLa , Humanos , Cinética , Ligantes , Microscopia de Fluorescência , Mitose , Domínios Proteicos , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química
6.
PLoS One ; 15(2): e0228036, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32015565

RESUMO

Atomic Force Microscopy was utilized to study the morphology of Gag, ΨRNA, and their binding complexes with lipids in a solution environment with 0.1Å vertical and 1nm lateral resolution. TARpolyA RNA was used as a RNA control. The lipid used was phospha-tidylinositol-(4,5)-bisphosphate (PI(4,5)P2). The morphology of specific complexes Gag-ΨRNA, Gag-TARpolyA RNA, Gag-PI(4,5)P2 and PI(4,5)P2-ΨRNA-Gag were studied. They were imaged on either positively or negatively charged mica substrates depending on the net charges carried. Gag and its complexes consist of monomers, dimers and tetramers, which was confirmed by gel electrophoresis. The addition of specific ΨRNA to Gag is found to increase Gag multimerization. Non-specific TARpolyA RNA was found not to lead to an increase in Gag multimerization. The addition PI(4,5)P2 to Gag increases Gag multimerization, but to a lesser extent than ΨRNA. When both ΨRNA and PI(4,5)P2 are present Gag undergoes comformational changes and an even higher degree of multimerization.


Assuntos
Infecções por HIV/genética , HIV-1/genética , RNA Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/ultraestrutura , Membrana Celular/química , Membrana Celular/genética , Infecções por HIV/virologia , Soropositividade para HIV , HIV-1/química , HIV-1/patogenicidade , Humanos , Lipídeos/química , Microscopia de Força Atômica , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Fosfatidilinositol 4,5-Difosfato/química , Ligação Proteica , Multimerização Proteica/genética , RNA Viral/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
7.
Nat Commun ; 11(1): 876, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054835

RESUMO

Cryo electron tomography with subsequent subtomogram averaging is a powerful technique to structurally analyze macromolecular complexes in their native context. Although close to atomic resolution in principle can be obtained, it is not clear how individual experimental parameters contribute to the attainable resolution. Here, we have used immature HIV-1 lattice as a benchmarking sample to optimize the attainable resolution for subtomogram averaging. We systematically tested various experimental parameters such as the order of projections, different angular increments and the use of the Volta phase plate. We find that although any of the prominently used acquisition schemes is sufficient to obtain subnanometer resolution, dose-symmetric acquisition provides considerably better outcome. We discuss our findings in order to provide guidance for data acquisition. Our data is publicly available and might be used to further develop processing routines.


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Benchmarking , Microscopia Crioeletrônica/normas , Bases de Dados Factuais , Tomografia com Microscopia Eletrônica/normas , HIV-1/química , HIV-1/ultraestrutura , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Biologia Molecular/métodos , Biologia Molecular/normas , Vírion/química , Vírion/ultraestrutura
8.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941772

RESUMO

Extensive studies with subtype A BG505-derived HIV envelope glycoprotein (Env) immunogens have revealed that the dominant autologous neutralizing epitope in rabbits is located in an exposed region of the heavily glycosylated trimer that lacks potential N-linked glycosylation sites at positions 230, 241, and 289. The Env derived from B41, a subtype B virus, shares a glycan hole centered on positions 230 and 289. To test whether broader neutralization to the common glycan hole can be achieved, we immunized rabbits with B41 SOSIP (gp120-gp41 disulfide [SOS] with an isoleucine-to-proline mutation [IP] in gp41) alone, as well as B41 and BG505 coimmunization. We isolated autologous neutralizing antibodies (nAbs) and described their structure in complex with the B41 Env. Our data suggest that distinct autologous nAb lineages are induced by BG505 and B41 immunogens, even when both were administered together. In contrast to previously described BG505 glycan hole antibodies, the B41-specific nAbs accommodate the >97% conserved N241 glycan, which is present in B41. Single-particle cryo-electron microscopy studies confirmed that B41- and BG505-specific nAbs bind to overlapping glycan hole epitopes. We then used our high-resolution data to guide mutations in the BG505 glycan hole epitope in an attempt to broaden the reactivity of a B41-specific nAb, but we recovered only partial binding. Our data demonstrate that the lack of cross-reactivity in glycan hole antibodies is due to amino acid differences within the epitope, and our attempts to rationally design cross-reactive trimers resulted in only limited success. Thus, even for the immunodominant glycan hole shared between BG505 and B41, the prospect of designing prime-boost immunogens remains difficult.IMPORTANCE A glycan hole is one of the most dominant autologous neutralizing epitopes targeted on BG505 and B41 SOSIP trimer-immunized rabbits. Our high-resolution cryo-electron microscopy (cryoEM) studies of B41 in complex with a B41-specific antibody complex elucidate the molecular basis of this strain-specific glycan hole response. We conclude that even for the immunodominant glycan hole shared between BG505 and B41, the prospect of designing prime-boost immunogens remains difficult.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Anti-HIV/química , HIV-1/química , Polissacarídeos/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Animais , Microscopia Crioeletrônica , Glicosilação , Células HEK293 , Humanos , Epitopos Imunodominantes/genética , Interferometria , Testes de Neutralização , Conformação Proteica , Coelhos , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
9.
Biochim Biophys Acta Biomembr ; 1862(2): 183149, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816324

RESUMO

Viral protein R (Vpr) is a small accessory protein of 96 amino acids that is present in Human and simian immunodeficiency viruses. Among the very different properties that Vpr possesses we can find cell penetrating capabilities. Based on this and on its capacity to interact with nucleic acids we previously investigated the DNA transfection properties of Vpr and subfragments thereof. We found that fragments of the C-terminal helical domain of Vpr are able to deliver efficiently plasmid DNA into different cell lines. As the amphipathic helix may play a role in the interactions with membranes, we investigated whether insertion of a proline residue in the α-helix modifies the transfection properties of Vpr. Unexpectedly, we found that the resulting Vpr55-82 Pro70 peptide was even more efficient than wild type Vpr55-82 in the gene delivery assays. Using circular dichroism, light scattering and solid-state NMR techniques, we characterized the secondary structure and interactions of Vpr and several mutants with model membranes. A model is proposed where the proline shifts the dissociation equilibrium of the peptide-cargo complex and thereby its endosomal release.


Assuntos
Peptídeos Penetradores de Células/química , Técnicas de Transferência de Genes , Bicamadas Lipídicas/química , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/química , Substituição de Aminoácidos , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Células HEK293 , HIV-1/química , Humanos , Isoleucina/química , Isoleucina/genética , Prolina/química , Prolina/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo
10.
J Exp Med ; 217(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31704807

RESUMO

Well-ordered HIV-1 envelope glycoprotein (Env) trimers are prioritized for clinical evaluation, and there is a need for an improved understanding about how elicited B cell responses evolve following immunization. To accomplish this, we prime-boosted rhesus macaques with clade C NFL trimers and identified 180 unique Ab lineages from ∼1,000 single-sorted Env-specific memory B cells. We traced all lineages in high-throughput heavy chain (HC) repertoire (Rep-seq) data generated from multiple immune compartments and time points and expressed several as monoclonal Abs (mAbs). Our results revealed broad dissemination and high levels of somatic hypermutation (SHM) of most lineages, including tier 2 virus neutralizing lineages, following boosting. SHM was highest in the Ab complementarity determining regions (CDRs) but also surprisingly high in the framework regions (FRs), especially FR3. Our results demonstrate the capacity of the immune system to affinity-mature large numbers of Env-specific B cell lineages simultaneously, supporting the use of regimens consisting of repeated boosts to improve each Ab, even those belonging to less expanded lineages.


Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos B/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vacinação , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Regiões Determinantes de Complementaridade/genética , Feminino , Anticorpos Anti-HIV/imunologia , Infecções por HIV/virologia , HIV-1/química , Sequenciamento de Nucleotídeos em Larga Escala , Cadeias Pesadas de Imunoglobulinas/genética , Macaca mulatta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Célula Única , Hipermutação Somática de Imunoglobulina
11.
Nat Struct Mol Biol ; 26(12): 1167-1175, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31792452

RESUMO

The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein, a (gp120-gp41)3 trimer, mediates fusion of viral and host cell membranes after gp120 binding to host receptor CD4. Receptor binding triggers conformational changes allowing coreceptor (CCR5) recognition through CCR5's tyrosine-sulfated amino (N) terminus, release of the gp41 fusion peptide and fusion. We present 3.3 Å and 3.5 Å cryo-EM structures of E51, a tyrosine-sulfated coreceptor-mimicking antibody, complexed with a CD4-bound open HIV-1 native-like Env trimer. Two classes of asymmetric Env interact with E51, revealing tyrosine-sulfated interactions with gp120 mimicking CCR5 interactions, and two conformations of gp120-gp41 protomers (A and B protomers in AAB and ABB trimers) that differ in their degree of CD4-induced trimer opening and induction of changes to the fusion peptide. By integrating the new structural information with previous closed and open envelope trimer structures, we modeled the order of conformational changes on the path to coreceptor binding site exposure and subsequent viral-host cell membrane fusion.


Assuntos
Anticorpos/química , Antígenos CD4/química , Proteína gp120 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/química , HIV-1/química , Anticorpos/metabolismo , Reações Antígeno-Anticorpo , Sítios de Ligação , Antígenos CD4/metabolismo , Antígenos CD4/ultraestrutura , Microscopia Crioeletrônica , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp120 do Envelope de HIV/ultraestrutura , Proteína gp41 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/ultraestrutura , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Receptores CCR5/imunologia , Tirosina/análogos & derivados , Tirosina/química
12.
Nat Struct Mol Biol ; 26(12): 1176-1183, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31792451

RESUMO

HIV-1 virion infectivity factor (Vif) promotes degradation of the antiviral APOBEC3 (A3) proteins through the host ubiquitin-proteasome pathway to enable viral immune evasion. Disrupting Vif-A3 interactions to reinstate the A3-catalyzed suppression of human immunodeficiency virus type 1 (HIV-1) replication is a potential approach for antiviral therapeutics. However, the molecular mechanisms by which Vif recognizes A3 proteins remain elusive. Here we report a cryo-EM structure of the Vif-targeted C-terminal domain of human A3F in complex with HIV-1 Vif and the cellular cofactor core-binding factor beta (CBFß) at 3.9-Å resolution. The structure shows that Vif and CBFß form a platform to recruit A3F, revealing a direct A3F-recruiting role of CBFß beyond Vif stabilization, and captures multiple independent A3F-Vif interfaces. Together with our biochemical and cellular studies, our structural findings establish the molecular determinants that are critical for Vif-mediated neutralization of A3F and provide a comprehensive framework of how HIV-1 Vif hijacks the host protein degradation machinery to counteract viral restriction by A3F.


Assuntos
Citosina Desaminase/química , HIV-1/química , Produtos do Gene vif do Vírus da Imunodeficiência Humana/química , Subunidade beta de Fator de Ligação ao Core/química , Microscopia Crioeletrônica , Citosina Desaminase/antagonistas & inibidores , Citosina Desaminase/ultraestrutura , Humanos , Evasão da Resposta Imune , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Proteólise , Relação Estrutura-Atividade , Produtos do Gene vif do Vírus da Imunodeficiência Humana/farmacologia , Produtos do Gene vif do Vírus da Imunodeficiência Humana/ultraestrutura
13.
Proc Natl Acad Sci U S A ; 116(50): 25269-25277, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31757854

RESUMO

The HIV-1 envelope glycoprotein (Env) is sparsely incorporated onto assembling virus particles on the host cell plasma membrane in order for the virus to balance infectivity and evade the immune response. Env becomes trapped in a nascent particle on encounter with the polymeric viral protein Gag, which forms a dense protein lattice on the inner leaflet of the plasma membrane. While Env incorporation efficiency is readily measured biochemically from released particles, very little is known about the spatiotemporal dynamics of Env trapping events. Herein, we demonstrate, via high-resolution single-molecule tracking, that retention of Env trimers within single virus assembly sites requires the Env cytoplasmic tail (CT) and the L12 residue in the matrix (MA) domain of Gag but does not require curvature of the viral lattice. We further demonstrate that Env trimers are confined to subviral regions of a budding Gag lattice, supporting a model where direct interactions and/or steric corralling between the Env-CT and a lattice of MA trimers promote Env trapping and infectious HIV-1 assembly.


Assuntos
HIV-1/fisiologia , Montagem de Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Membrana Celular/virologia , Infecções por HIV/virologia , HIV-1/química , HIV-1/genética , Humanos , Ligação Proteica , Domínios Proteicos , Imagem Individual de Molécula , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
14.
PLoS One ; 14(11): e0224965, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31714942

RESUMO

Since its discovery in the early 1980s, there has been significant progress in understanding the biology of type 1 human immunodeficiency virus (HIV-1). Structural biologists have made tremendous contributions to this challenge, guiding the development of current therapeutic strategies. Despite our efforts, there are unresolved structural features of the virus and consequently, significant knowledge gaps in our understanding. The superstructure of the HIV-1 matrix (MA) shell has not been elucidated. Evidence by various high-resolution microscopy techniques support a model composed of MA trimers arranged in a hexameric configuration consisting of 6 MA trimers forming a hexagon. In this manuscript we review the mathematical limitations of this model and propose a new model consisting of a 6-lune hosohedra structure, which aligns with available structural evidence. We used geometric and rotational matrix computation methods to construct our model and predict a new mechanism for viral entry that explains the increase in particle size observed during CD4 receptor engagement and the most common HIV-1 ellipsoidal shapes observed in cryo-EM tomograms. A better understanding of the HIV-1 MA shell structure is a key step towards better models for viral assembly, maturation and entry. Our new model will facilitate efforts to improve understanding of the biology of HIV-1.


Assuntos
HIV-1/química , HIV-1/fisiologia , Modelos Moleculares , Proteínas da Matriz Viral/química , Peptídeos/química , Vírion/química , Montagem de Vírus
15.
J Chem Phys ; 151(16): 165101, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31675878

RESUMO

As an extremely common structural motif, RNA hairpins with bulge loops [e.g., the human immunodeficiency virus type 1 (HIV-1) transactivation response (TAR) RNA] can play essential roles in normal cellular processes by binding to proteins and small ligands, which could be very dependent on their three-dimensional (3D) structures and stability. Although the structures and conformational dynamics of the HIV-1 TAR RNA have been extensively studied, there are few investigations on the thermodynamic stability of the TAR RNA, especially in ion solutions, and the existing studies also have some divergence on the unfolding process of the RNA. Here, we employed our previously developed coarse-grained model with implicit salt to predict the 3D structure, stability, and unfolding pathway for the HIV-1 TAR RNA over a wide range of ion concentrations. As compared with the extensive experimental/theoretical results, the present model can give reliable predictions on the 3D structure stability of the TAR RNA from the sequence. Based on the predictions, our further comprehensive analyses on the stability of the TAR RNA as well as its variants revealed that the unfolding pathway of an RNA hairpin with a bulge loop is mainly determined by the relative stability between different states (folded state, intermediate state, and unfolded state) and the strength of the coaxial stacking between two stems in folded structures, both of which can be apparently modulated by the ion concentrations as well as the sequences.


Assuntos
HIV-1/química , Conformação de Ácido Nucleico , RNA Viral/química , Íons/química , Modelos Moleculares , Soluções
16.
Proc Natl Acad Sci U S A ; 116(45): 22556-22566, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31624123

RESUMO

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.


Assuntos
Membrana Celular/virologia , Proteína gp41 do Envelope de HIV/química , HIV-1/imunologia , Anticorpos Neutralizantes/imunologia , Membrana Celular/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/química , HIV-1/genética , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/imunologia , Domínios Proteicos
17.
Microb Pathog ; 137: 103791, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31606417

RESUMO

Till now, AIDS, caused by the human immunodeficiency virus (HIV) is still a severe health problem worldwide. It weakens the immune system by targeting the T-helper cells. Specifically, the severity of the pandemic HIV-1 makes the emergence of an enduring effective vaccine against HIV-1. Therefore, we have applied a series of immunoinformatics approaches to the four conserved domains of HIV-1 integrase (IN) proteins to design an effective multi-epitope based subunit vaccine which might induce a competent immunity against HIV-1. Therefore, we have selected three peptide fragments that contained all overlapping epitopes (35 CD4+, 8 CD8+ T-cell epitopes, and 3 B-cell epitopes) where the epitopes had a high conservancy score. The cumulative population coverage for combined CD8+ and CD4+ T-cell epitopes and their respective HLA-alleles were found as 98.03% in the world which is also followed by East Asia (96.24%), South Asia (96.31%), North Africa (96.14%), North America (98.99%), and Europe (98.80%). The proposed vaccine composed by an adjuvant (ß-defensin) at the N-terminal site of the vaccine constructs and three peptide fragments where the adjuvant was fused by EAAAK linker and the peptide fragments were fused by GPGPG linker. The designed final vaccine construct (length: 159 amino acid) was found to be antigenic and non-allergic, which indicates its safety. The vaccine construct was found as good antigenic, stable, higher thermostable, and hydrophilic in nature. The codon adaptation and in silico cloning ensured the high expression rate of the vaccine constructs in E. coli K12 with CAI value of 1.0. Finally, the binding affinity of the vaccine constructs with the immune receptor TLR3 was confirmed by the lowest energy score of -1026.8 evaluated by molecular docking. However, the proposed in silico vaccine construct needs experimental validation for assuring the safety and immunogenicity profile which will ensure an active immunity against HIV-1.


Assuntos
Vacinas contra a AIDS/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Infecções por HIV/imunologia , Integrase de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/genética , Sequência de Aminoácidos , Ásia , Biologia Computacional , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Europa (Continente) , Infecções por HIV/genética , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Integrase de HIV/química , Integrase de HIV/genética , HIV-1/química , HIV-1/genética , Humanos , Simulação de Acoplamento Molecular , Domínios Proteicos , Vacinas de Subunidades/química , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia
18.
ACS Appl Mater Interfaces ; 11(38): 34586-34594, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31483592

RESUMO

The human immunodeficiency virus 1 (HIV-1) capsid serves as a binding platform for proteins and small molecules from the host cell that regulate various steps in the virus life cycle. However, there are currently no quantitative methods that use assembled capsid lattices to measure host-pathogen interaction dynamics. Here we developed a single-molecule fluorescence biosensor using self-assembled capsid tubes as biorecognition elements and imaged capsid binders using total internal reflection fluorescence microscopy in a microfluidic setup. The method is highly sensitive in its ability to observe and quantify binding, to obtain dissociation constants, and to extract kinetics with an extended application of using more complex analytes that can accelerate characterization of novel capsid binders.


Assuntos
Técnicas Biossensoriais , Capsídeo , HIV-1 , Dispositivos Lab-On-A-Chip , Capsídeo/química , Capsídeo/metabolismo , HIV-1/química , HIV-1/metabolismo , Humanos , Microscopia de Fluorescência
19.
PLoS Comput Biol ; 15(9): e1007345, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31545786

RESUMO

HIV-1 replicates via a low-fidelity polymerase with a high mutation rate; strong conservation of individual nucleotides is highly indicative of the presence of critical structural or functional properties. Identifying such conservation can reveal novel insights into viral behaviour. We analysed 3651 publicly available sequences for the presence of nucleic acid conservation beyond that required by amino acid constraints, using a novel scale-free method that identifies regions of outlying score together with a codon scoring algorithm. Sequences with outlying score were further analysed using an algorithm for producing local RNA folds whilst accounting for alignment properties. 11 different conserved regions were identified, some corresponding to well-known cis-acting functions of the HIV-1 genome but also others whose conservation has not previously been noted. We identify rational causes for many of these, including cis functions, possible additional reading frame usage, a plausible mechanism by which the central polypurine tract primes second-strand DNA synthesis and a conformational stabilising function of a region at the 5' end of env.


Assuntos
Sequência Conservada/genética , Genoma Viral/genética , HIV-1/genética , Algoritmos , Códon/genética , Biologia Computacional , HIV-1/química , HIV-1/ultraestrutura , Modelos Genéticos , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , RNA Viral/ultraestrutura
20.
Adv Virus Res ; 105: 239-273, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31522706

RESUMO

Single-molecule Förster resonance energy transfer (smFRET) imaging has emerged as a powerful tool to probe conformational dynamics of viral proteins, identify novel structural intermediates that are hiding in averaging population-based measurements, permit access to the energetics of transitions and as such to the precise molecular mechanisms of viral replication. One strength of smFRET is the capability of characterizing biological molecules in their fully hydrated/native state, which are not necessarily available to other structural methods. Elegant experimental design for physiologically relevant conditions, such as intact virions, has permitted the detection of previously unknown conformational states of viral glycoproteins, revealed asymmetric intermediates, and allowed access to the real-time imaging of conformational changes during viral fusion. As more laboratories are applying smFRET, our understanding of the molecular mechanisms and the dynamic nature of viral proteins throughout the virus life cycle are predicted to improve and assist the development of novel antiviral therapies and vaccine design.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , HIV-1/química , HIV-1/fisiologia , Conformação Proteica , Internalização do Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
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