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1.
Nat Commun ; 10(1): 2898, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263112

RESUMO

The HIV-1 envelope (Env) is the target for neutralizing antibodies and exists on the surface of virions in open or closed conformations. Difficult-to-neutralize viruses (tier 2) express Env in a closed conformation antigenic for broadly neutralizing antibodies (bnAbs) but not for third variable region (V3) antibodies. Here we show that select V3 macaque antibodies elicited by Env vaccination can neutralize 26% of otherwise tier 2 HIV-1 isolates in standardized virus panels. The V3 antibodies only bound to Env in its open conformation. Thus, Envs on tier 2 viruses sample a state where the V3 loop is not in its closed conformation position. Envelope second variable region length, glycosylation sites and V3 amino acids were signatures of neutralization sensitivity. This study determined that open conformations of Env with V3 exposed are present on a subset of otherwise neutralization-resistant virions, therefore neutralization of tier 2 HIV-1 does not always indicate bnAb induction.


Assuntos
Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Motivos de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Glicosilação , Infecções por HIV/virologia , HIV-1/química , HIV-1/genética , Humanos , Macaca mulatta , Testes de Neutralização , Conformação Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/química
2.
Soft Matter ; 15(22): 4525-4540, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31099376

RESUMO

The complex-type glycan shields of eukaryotic cells have a core layer of mannose residues buried under tiers of sugars that end with sialic acid (SA) residues. We investigate if the self-latching of mannose residues, earlier reported in pure monolayer studies, also manifests in the setting of a complex-type glycan shield. Would distal SA residues impede access to the mannose core? The interactions of mannobiose-, SA-, and lactose-coated probes with the complex-type VSV-G glycan shield on an HIV pseudovirus were studied with force-spectroscopy and gold-nanoparticle solutions. In force spectroscopy, the sugar probes can be forced to sample the depths of the glycan shield, whereas with sugar-coated nanoparticles, only interactions permitted by freely-diffusive contact occur. Deep-indentation mechanics was performed to verify the inferred structure of the engineered virus and to isolate the glycan shield layer for subsequent interaction studies. The adhesion between the sugar-probes and complex-type glycan shield was deconvoluted by comparing against the cross- and self- adhesions between the sugars in pure monolayers. Results from complementing systems were consistent with mannobiose-coated probes latching to the mannose core in the glycan shield, unhindered by the SA and distal sugars, with a short-range 'brittle' release of adhesion resulting in tightly coated viruses. SA-Coated probes, however, adhere to the terminal SA layer of a glycan shield with long-range and mechanically 'tough' adhesions resulting in large-scale virus aggregation. Lactose-coated probes exhibit ill-defined adherence to sialic residues. The selection and positioning of sugars within a glycan shield can influence how carbohydrate surfaces of different composition adhere.


Assuntos
HIV-1/química , Manose/química , Glicoproteínas de Membrana/química , Ácido N-Acetilneuramínico/química , Proteínas do Envelope Viral/química , Células HEK293 , HIV-1/genética , HIV-1/metabolismo , Humanos , Manose/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Nanopartículas Metálicas/química , Ácido N-Acetilneuramínico/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
3.
Nucleic Acids Res ; 47(7): 3739-3751, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30993347

RESUMO

Investigating the dynamics of structural elements in functional RNAs is important to better understand their mechanism and for engineering RNAs with novel functions. Previously, we performed rational engineering studies with the Varkud satellite (VS) ribozyme and switched its specificity toward non-natural hairpin substrates through modification of a critical kissing-loop interaction (KLI). We identified functional VS ribozyme variants with surrogate KLIs (ribosomal RNA L88/L22 and human immunodeficiency virus-1 TAR/TAR*), but they displayed ∼100-fold lower cleavage activity. Here, we characterized the dynamics of KLIs to correlate dynamic properties with function and improve the activity of designer ribozymes. Using temperature replica exchange molecular dynamics, we determined that the natural KLI in the VS ribozyme supports conformational sampling of its closed and active state, whereas the surrogate KLIs display more restricted motions. Based on in vitro selection, the cleavage activity of a VS ribozyme variant with the TAR/TAR* KLI could be markedly improved by partly destabilizing the KLI but increasing conformation sampling. We formulated a mechanistic model for substrate binding in which the KLI dynamics contribute to formation of the active site. Our model supports the modular nature of RNA in which subdomain structure and dynamics contribute to define the thermodynamics and kinetics relevant to RNA function.


Assuntos
Endorribonucleases/química , HIV-1/química , RNA Catalítico/química , Proteínas de Ligação a RNA/química , RNA/química , Sítios de Ligação , Endorribonucleases/genética , Genes de RNAr/genética , HIV-1/genética , Modelos Moleculares , Conformação de Ácido Nucleico , RNA/genética , RNA Catalítico/genética , RNA não Traduzido/química , RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Termodinâmica
4.
Int J Mol Sci ; 20(7)2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987231

RESUMO

The Human immunodeficiency virus-1 (HIV-1) matrix (MA) domain is involved in the highly regulated assembly process of the virus particles that occur at the host cell's plasma membrane. High-resolution structures of the MA domain determined using cryo X-ray crystallography have provided initial insights into the possible steps in the viral assembly process. However, these structural studies have relied on large and frozen crystals in order to reduce radiation damage caused by the intense X-rays. Here, we report the first X-ray free-electron laser (XFEL) study of the HIV-1 MA domain's interaction with inositol hexaphosphate (IP6), a phospholipid headgroup mimic. We also describe the purification, characterization and microcrystallization of two MA crystal forms obtained in the presence of IP6. In addition, we describe the capabilities of serial femtosecond X-ray crystallography (SFX) using an XFEL to elucidate the diffraction data of MA-IP6 complex microcrystals in liquid suspension at ambient temperature. Two different microcrystal forms of the MA-IP6 complex both diffracted to beyond 3.5 Å resolution, demonstrating the feasibility of using SFX to study the complexes of MA domain of HIV-1 Gag polyprotein with IP6 at near-physiological temperatures. Further optimization of the experimental and data analysis procedures will lead to better understanding of the MA domain of HIV-1 Gag and IP6 interaction at high resolution and will provide basis for optimization of the lead compounds for efficient inhibition of the Gag protein recruitment to the plasma membrane prior to virion formation.


Assuntos
HIV-1/química , Temperatura Ambiente , Difração de Raios X , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Cristalização , Modelos Moleculares , Domínios Proteicos , Fatores de Tempo , Vírion/química
5.
Neurochem Res ; 44(7): 1636-1652, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31006091

RESUMO

HIV-1 gp120, an important subunit of the envelope spikes that decorate the surface of virions, is known to play a vital role in neuronal injury during HIV-1-associated neurocognitive disorder (HAND), although the pathological mechanism is not fully understood. Our previous studies have suggested that the V3 loop of HIV-1 gp120 (HIV-1 gp120 V3 loop) can induce neuronal apoptosis in the hippocampus, resulting in impairment in spatial learning and memory in Sprague-Dawley (SD) rats. In this study, we demonstrated that autophagy was significantly increased in rat primary hippocampal neurons in response to treatment of HIV-1 gp120 V3 loop. Importantly, HIV-1 gp120 V3 loop-induced autophagy played a dual role in the cell survival and death. An increase in autophagy for a short period inhibited apoptosis of neurons, while persistent autophagy over an extended period of time played a detrimental role by augmenting the apoptotic cascade in rat primary hippocampal neurons. In addition, we found that the HIV-1 gp120 V3 loop induced autophagy via AMPK/mTOR-dependent and calpain/mTOR-independent pathways, and the ERK/mTOR pathway plays a partial role. These findings provide evidence that HIV-1-induced autophagy plays a dual role in the survival and apoptosis of the primary rat hippocampal neurons and persistent autophagy may contribute to the pathogenesis of HAND, and autophagy modulation may represent a potential therapeutic strategy for reducing neuronal damage in HAND.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/farmacologia , HIV-1/química , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Calpaína/antagonistas & inibidores , Calpaína/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Flavonoides/farmacologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/toxicidade , Hipocampo/patologia , Masculino , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Inibidores de Proteínas Quinases/farmacologia , Ratos Sprague-Dawley
6.
Biosens Bioelectron ; 133: 55-63, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30909013

RESUMO

A bimetallic NiCo-based metal-organic framework (NiCo-MOF) was pyrolyzed into a novel composite comprising NiCo2O4 spinel, CoO, and metallic Co/Ni nanoparticles embedded with carbon nanotubes at high temperature 700 °C under N2 atmosphere (represented by NiCo2O4/CoO@CNTs), whereas organic ligands were almost decomposed in H2 atmosphere, leading to absence of carbon nanotubes (denoted by NiCo2O4/CoO). The feasibility of using the composite as efficient bioplatform for immobilizing the probe DNA of human immune deficiency virus-1 (HIV-1) in the electrochemical detection of the HIV-1 DNA was explored. Compared with the pristine NiCo-MOF and NiCo2O4/CoO, the NiCo2O4/CoO@CNTs composite exhibited high electrochemical activity, good biocompatibility, and strong bioaffinity toward the probe DNA. Electrochemical measurements demonstrated that the NiCo2O4/CoO@CNTs-based bioassay displayed superior sensing performances, giving an ultralow detection limit of 16.7 fM toward HIV-1 DNA over the linear range of 0.1 pM to 20 nM; possessing high selectivity even against noncomplementary and two-base mismatch sequences; exhibiting good stability, reproducibility, repeatability and applicability in the application of detecting human serum samples. Thus, the present strategy can broaden the application of MOFs and their derivatives in various fields of biosensing.


Assuntos
Técnicas Biossensoriais , DNA Viral/isolamento & purificação , HIV-1/isolamento & purificação , Nanotubos de Carbono/química , Cobalto/química , DNA Viral/química , Glucose , HIV-1/química , Humanos , Nanopartículas Metálicas/química , Estruturas Metalorgânicas/química , Níquel/química , Óxidos/química
7.
J Biol Chem ; 294(14): 5616-5631, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30728245

RESUMO

A successful HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) that target the envelope glycoprotein (Env) spike on the virus. Native-like recombinant Env trimers of the SOSIP design now serve as a platform for achieving this challenging goal. However, SOSIP trimers usually do not bind efficiently to the inferred germline precursors of bNAbs (gl-bNAbs). We hypothesized that the inherent flexibilities of the V1 and V2 variable loops in the Env trimer contribute to the poor recognition of gl-bNAb epitopes at the trimer apex that extensively involve V2 residues. To reduce local V2 flexibility and improve the binding of V2-dependent bNAbs and gl-bNAbs, we designed BG505 SOSIP.664 trimer variants containing newly created disulfide bonds intended to stabilize the V2 loop in an optimally antigenic configuration. The first variant, I184C/E190C, contained a new disulfide bond within the V2 loop, whereas the second variant, E153C/R178C, had a new disulfide bond that cross-linked V2 and V1. The resulting engineered native-like trimer variants were both more reactive with and were neutralized by V2 bNAbs and gl-bNAbs, a finding that may be valuable in the design of germline targeting and boosting trimer immunogens to create an antigenic conformation optimal for HIV vaccine development.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Multimerização Proteica/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/química , Anticorpos Neutralizantes/química , Epitopos/química , Anticorpos Anti-HIV/química , HIV-1/química , Estrutura Secundária de Proteína
8.
Colloids Surf B Biointerfaces ; 177: 77-93, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30711762

RESUMO

Peptide epitopes have been widely used to develop synthetic vaccines and immunotherapies. However, peptide epitopes may exhibit poor absorption or immunogenicity due to their low molecular weights. Conversely, fourth-generation polyamidoamine (G4-PAMAM) dendrimers are nonimmunogenic and relatively nontoxic synthetic nanoparticles that have been used as adjuvants and nanocarriers of small peptides and to improve nasal absorption. Based on this information, we hypothesized that the combination of intranasal immunization and G4-PAMAM dendrimers would be useful for enhancing the antibody responses of HIV-1 gp120 peptide epitopes. Therefore, we first used structural data, peptide epitope predictors and docking and MD simulations on MHC-II to identify two peptide epitopes on the CD4 binding site of HIV-1 gp120. The formation of G4-PAMAM-peptide complexes was evaluated in silico (molecular docking studies using different G4-PAMAM conformations retrieved from MD simulations as well as the MMGBSA approach) and validated experimentally (electrophoresis, 1H NMR and cryo-TEM). Next, the G4-PAMAM dendrimer-peptide complexes were administered intranasally to groups of female BALB/cJ mice. The results showed that both peptides were immunogenic at the systemic and mucosal levels (nasal and vaginal), and G4-PAMAM dendrimer-peptide complexes improved IgG and IgA responses in serum and nasal washes. Thus, G4-PAMAM dendrimers have potential for use as adjuvants and nanocarriers of peptides.


Assuntos
Simulação por Computador , Dendrímeros/química , Proteína gp120 do Envelope de HIV/química , HIV-1/química , HIV-1/imunologia , Modelos Moleculares , Nylons/química , Peptídeos/química , Peptídeos/imunologia , Animais , Feminino , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/genética
9.
Biochem Biophys Res Commun ; 509(2): 564-569, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30600181

RESUMO

Human immunodeficiency virus type-1 (HIV-1) transactivator of transcription (Tat) is an intrinsically disordered protein that exerts multiple functions, including activation of HIV-1 replication and induction of T-cell apoptosis and cytokine secretion via zinc binding and cellular uptake by endocytosis. However, the effects of zinc and endosomal low pH on the structure of isolated Tat protein are poorly understood. Here, we purified a monomeric zinc-bound Tat and studied its structure and acid denaturation by circular dichroism, NMR, and small-angle X-ray scattering. We found that at pH 7, the zinc-bound Tat was in a pre-molten globule state; it exhibited largely disordered conformations with residual helices and was slightly more compact than the fully unfolded states that were observed at pH 4 or in the zinc-free form. Moreover, acid-induced unfolding transitions in secondary structure and molecular size occurred at different pH ranges, indicating the presence of an expanded and helical intermediate at pH ∼6. Taken together, the extent of structural disorder in the intrinsically disordered Tat protein is highly sensitive to zinc and pH, suggesting that zinc binding and pH affect Tat structures and thereby control the versatile functions of Tat.


Assuntos
Infecções por HIV/virologia , HIV-1/metabolismo , Zinco/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , HIV-1/química , Humanos , Concentração de Íons de Hidrogênio , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química
10.
Biochim Biophys Acta Gen Subj ; 1863(1): 13-24, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30248376

RESUMO

BACKGROUND: HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships. METHODS: We used: biophysical techniques (circular dichroism (CD), nuclear magnetic resonance (NMR) and thermal/GuHCL denaturation) to study protein conformation and stability; Surface plasmon resonance (SPR) to study interactions; Western blot to investigate signaling pathways; and Colony Formation and Soft Agar assays to study B cell proliferation and clonogenicity. RESULTS: By forcing the formation of a disulfide bridge between Cys residues at positions 57 and 87 we obtained a destabilized p17 capable of promoting B cell proliferation. This finding prompted us to dissect refp17 to identify the functional epitope. A synthetic peptide (F1) spanning from amino acid (aa) 2 to 21 was found to activate Akt and promote B cell proliferation and clonogenicity. Three positively charged aa (Arg15, Lys18 and Arg20) proved critical for sustaining the proliferative activity of both F1 and HIV-NHL-derived vp17s. Lack of any interaction of F1 with the known refp17 receptors suggests an alternate one involved in cell proliferation. CONCLUSIONS: The molecular reasons for the proliferative activity of vp17s, compared to refp17, relies on the exposure of a functional epitope capable of activating Akt. GENERAL SIGNIFICANCE: Our findings pave the way for identifying the receptor(s) responsible for B cell proliferation and offer new opportunities to identify novel treatment strategies in combating HIV-related NHL.


Assuntos
Linfócitos B/virologia , Antígenos HIV/química , Infecções por HIV/imunologia , HIV-1/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Linfócitos B/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Dissulfetos/química , Epitopos/química , Humanos , Luz , Peptídeos/química , Conformação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/química , Espalhamento de Radiação , Transdução de Sinais , Ressonância de Plasmônio de Superfície
11.
Proc Natl Acad Sci U S A ; 115(38): E8892-E8899, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30185554

RESUMO

The membrane-proximal external region (MPER) of the HIV-1 envelope glycoprotein (Env) bears epitopes of broadly neutralizing antibodies (bnAbs) from infected individuals; it is thus a potential vaccine target. We report an NMR structure of the MPER and its adjacent transmembrane domain in bicelles that mimic a lipid-bilayer membrane. The MPER lies largely outside the lipid bilayer. It folds into a threefold cluster, stabilized mainly by conserved hydrophobic residues and potentially by interaction with phospholipid headgroups. Antigenic analysis and comparison with published images from electron cryotomography of HIV-1 Env on the virion surface suggest that the structure may represent a prefusion conformation of the MPER, distinct from the fusion-intermediate state targeted by several well-studied bnAbs. Very slow bnAb binding indicates that infrequent fluctuations of the MPER structure give these antibodies occasional access to alternative conformations of MPER epitopes. Mutations in the MPER not only impede membrane fusion but also influence presentation of bnAb epitopes in other regions. These results suggest strategies for developing MPER-based vaccine candidates.


Assuntos
Antígenos HIV/química , HIV-1/química , Vírion/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Antígenos HIV/imunologia , HIV-1/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Fusão de Membrana , Domínios Proteicos , Vírion/imunologia
12.
Proc Natl Acad Sci U S A ; 115(33): E7854-E7862, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30061386

RESUMO

The transmission of HIV can be prevented by the application of neutralizing monoclonal antibodies and lectins. Traditional recombinant protein manufacturing platforms lack sufficient capacity and are too expensive for developing countries, which suffer the greatest disease burden. Plants offer an inexpensive and scalable alternative manufacturing platform that can produce multiple components in a single plant, which is important because multiple components are required to avoid the rapid emergence of HIV-1 strains resistant to single microbicides. Furthermore, crude extracts can be used directly for prophylaxis to avoid the massive costs of downstream processing and purification. We investigated whether rice could simultaneously produce three functional HIV-neutralizing proteins (the monoclonal antibody 2G12, and the lectins griffithsin and cyanovirin-N). Preliminary in vitro tests showed that the cocktail of three proteins bound to gp120 and achieved HIV-1 neutralization. Remarkably, when we mixed the components with crude extracts of wild-type rice endosperm, we observed enhanced binding to gp120 in vitro and synergistic neutralization when all three components were present. Extracts of transgenic plants expressing all three proteins also showed enhanced in vitro binding to gp120 and synergistic HIV-1 neutralization. Fractionation of the rice extracts suggested that the enhanced gp120 binding was dependent on rice proteins, primarily the globulin fraction. Therefore, the production of HIV-1 microbicides in rice may not only reduce costs compared to traditional platforms but may also provide functional benefits in terms of microbicidal potency.


Assuntos
Fármacos Anti-HIV , Anticorpos Monoclonais , Endosperma , Anticorpos Anti-HIV , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , HIV-1/química , Oryza , Plantas Geneticamente Modificadas , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Endosperma/química , Endosperma/genética , Endosperma/metabolismo , Anticorpos Anti-HIV/biossíntese , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Oryza/química , Oryza/genética , Oryza/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo
13.
Virology ; 523: 1-5, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30056211

RESUMO

The capsid-binding assay is an in vitro experiment used to determine whether cellular proteins interact with the HIV-1 core. In vitro assembled HIV-1 capsids recapitulate the surface of the HIV-1 core. The assay involves the incubation of in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes with the protein in question. Subsequently, the mixture is spun through a sucrose cushion using an ultracentrifuge, and the pellet is analyzed for the presence of the protein in question. Although this binding assay is reliable, it is labor intensive and does not contain washing steps. Here we have developed a simpler and faster assay to measure whether a cellular protein is binding to capsid. More importantly, this novel capsid-binding assay contains washing steps. In this assay, we took advantage of the HIV-1 capsid mutant A14C/E45C protein, which is stabilized by disulfide bonds, and is resistant to washing steps. We validated the reliability and specificity of this novel assay by testing the capsid binding ability of TRIMCyp, CPSF6 and MxB with their corresponding controls. Overall, this novel assay provides a reliable and fast methodology to search for novel capsid binders.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Bioensaio , Proteínas do Capsídeo/química , HIV-1/química , Proteínas de Membrana/química , Proteínas de Resistência a Myxovirus/química , Fatores de Poliadenilação e Clivagem de mRNA/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , HIV-1/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutação , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Nucleocapsídeo/química , Nucleocapsídeo/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
14.
J Virol ; 92(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29976675

RESUMO

We have previously reported that the CBD1 peptide (SLEQIWNNMTWMQWDK), corresponding to the consensus caveolin-1 binding domain in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41, elicits peptide-specific antibodies. Here, we have investigated the cellular immune response and the protective efficacy against a simian/human immunodeficiency virus (SHIV162P3) challenge. In addition to the CBD1 peptide, peptides overlapping the caveolin-binding-motif (CBM) (622IWNNMTWMQW631 or 622IWNNMTW628) were fused to a Gag-p24 T helper epitope for vaccination. All immunized cynomolgus macaques responded to a cocktail peptide immunization by inducing specific T cells and the production of high-titer CBD1/CBM peptide-specific antibodies. Six months after the fourth vaccine boost, six control and five vaccinated animals were challenged weekly by repeated exposure to SHIV162P3 via the mucosal rectal route. All control animals were infected after 1 to 3 challenges with SHIV, while among the five vaccinated monkeys, three became infected after a delay compared to control; one was infected after the eighth viral challenge, and one remained uninfected even after the ninth SHIV challenge. Immunized animals maintained a CD4 T cell count, and their central memory CD4 T cells were less depleted than in the control group. Furthermore, SHIV challenge stimulates antigen-specific memory T cell response in vaccinated macaques. Our results indicate that peptides derived from the CBM region can be immunogenic and provide protection against SHIV infection in cynomolgus monkeys.IMPORTANCE In HIV-1-producing cells, gp41 exists in a complexed form with caveolin-1, an interaction most probably mediated by the caveolin-1 binding motif. This sequence is highly conserved in every single HIV-1 isolate, thus suggesting that there is constant selective pressure to preserve this sequence for a specific function in the HIV infectious cycle. Consequently, the CBM sequence may represent the "Achilles' heel" of HIV-1 in the development of an efficient vaccine. Our results demonstrate that macaques immunized with the CBM-based peptides displayed a delay in the onset of viral infection and CD4 depletion, as well as a significant induction of antigen-specific memory T cell response, which is essential for the control of HIV/SIV infections. Finally, as HIV-infected individuals lack anti-CBM immune responses, CBM-based vaccines could have applications as a therapeutic vaccine in AIDS patients.


Assuntos
Vacinas contra a AIDS , Caveolina 1/química , Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Peptídeos/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Contagem de Linfócito CD4 , Caveolina 1/metabolismo , Proteína gp41 do Envelope de HIV/administração & dosagem , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Infecções por HIV/imunologia , HIV-1/química , HIV-1/genética , Humanos , Imunidade Celular , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Memória Imunológica , Macaca fascicularis , Peptídeos/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Linfócitos T/imunologia , Células Th1/imunologia , Vacinação
15.
Subcell Biochem ; 88: 189-210, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29900498

RESUMO

Integration of a DNA copy of the viral genome into host DNA is an essential step in the replication cycle of HIV-1 and other retroviruses and is an important therapeutic target for drugs. DNA integration is catalyzed by the viral integrase protein and proceeds through a series of stable nucleoprotein complexes of integrase, viral DNA ends and target DNA. These nucleoprotein complexes are collectively called intasomes. Retroviral intasomes undergo a series of transitions between initial formation and catalysis of the DNA cutting and joining steps of DNA integration. Intasomes, rather than free integrase protein, are the target of currently approved drugs that target HIV-1 DNA integration. High-resolution structures of HIV-1 intasomes are needed to understand their detailed mechanism of action and how HIV-1 may escape by developing resistance. Here, we focus on our current knowledge of the structure and function of HIV-1 intasomes, with reference to related systems as required to put this knowledge in context.


Assuntos
DNA Viral/metabolismo , HIV-1/fisiologia , Nucleoproteínas/metabolismo , Integração Viral/fisiologia , Animais , DNA Viral/genética , HIV-1/química , Humanos , Nucleoproteínas/química , Nucleoproteínas/genética , Relação Estrutura-Atividade
16.
Virology ; 519: 180-189, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29729526

RESUMO

The twin-cysteine motif (TCM) in the V2 loop region of gp120, identified in our previous report on the simian immunodeficiency virus mac239 (SIVmac239), is a conserved evolutionary element in all primate lentiviruses except for HIV-1 which has lost the TCM during cross-species transmission. In this study, we have further explored the TCM in other SIV and HIV-2 strains. Our data shows that strains from different evolutionary lineages have different phenotypes when the twin-cysteines are removed. In the SIVsm/HIV-2 lineage, removal of the twin-cysteines decreases envelope trimer stability, but in the SIVagm lineage, a blockage of gp160 processing is observed. Molecular modeling has confirmed that the twin-cysteines do form a disulfide bond in the gp120 subunit, which interacts with the V1 loop to stabilize the envelope trimer. Therefore, we hypothesize that if the TCM is added back to HIV-1, it will enhance envelope stability for vaccine immunogen design.


Assuntos
Motivos de Aminoácidos , Cisteína/química , Proteína gp120 do Envelope de HIV/química , HIV-1/química , HIV-2/química , Vírus da Imunodeficiência Símia/química , Proteínas do Envelope Viral/química , Vacinas contra a AIDS , Sequência de Aminoácidos , Animais , Linhagem Celular , Cisteína/genética , Desenho de Drogas , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , HIV-2/genética , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Vírus da Imunodeficiência Símia/genética , Proteínas do Envelope Viral/genética
17.
Viruses ; 10(5)2018 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-29772652

RESUMO

Simian-human immunodeficiency virus (SHIV) infection provides a relevant animal model to study HIV-1 neutralization breadth. With previously identified SHIVSF162P3N infected rhesus macaques that did or did not develop neutralization breadth, we characterized the transmitted/founder viruses and initial autologous/homologous neutralizing antibodies in these animals. The plasma viral load and blood CD4 count did not distinguish macaques with and without breadth, and only one tested homologous envelope clone revealed a trend for macaques with breadth to favor an early homologous response. In two macaques with breadth, GB40 and FF69, infected with uncloned SHIVSF162P3N, multiple viral variants were transmitted, and the transmitted variants were not equal in neutralization sensitivity. The targets of initial autologous neutralizing antibodies, arising between 10 and 20 weeks post infection, were mapped to N462 glycan and G460a in gp120 V5 in GB40 and FF69, respectively. Although it is unclear whether these targets are related to later neutralization breadth development, the G460a target but not N462 glycan appeared more common in macaques with breadth than those without. Longitudinal plasmas revealed 2⁻3 sequential waves of neutralizing antibodies in macaques with breadth, implicating that 3 sequential envelope variants, if not more, may be required for the broadening of HIV-1 neutralizing antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/sangue , Mapeamento de Epitopos , Epitopos/imunologia , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/química , HIV-1/química , Macaca mulatta , Modelos Moleculares , Testes de Neutralização , Sensibilidade e Especificidade , Alinhamento de Sequência , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Fatores de Tempo
18.
PLoS Pathog ; 14(5): e1006986, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29746590

RESUMO

Inducing broad spectrum neutralizing antibodies against challenging pathogens such as HIV-1 is a major vaccine design goal, but may be hindered by conformational instability within viral envelope glycoproteins (Env). Chemical cross-linking is widely used for vaccine antigen stabilization, but how this process affects structure, antigenicity and immunogenicity is poorly understood and its use remains entirely empirical. We have solved the first cryo-EM structure of a cross-linked vaccine antigen. The 4.2 Å structure of HIV-1 BG505 SOSIP soluble recombinant Env in complex with a CD4 binding site-specific broadly neutralizing antibody (bNAb) Fab fragment reveals how cross-linking affects key properties of the trimer. We observed density corresponding to highly specific glutaraldehyde (GLA) cross-links between gp120 monomers at the trimer apex and between gp120 and gp41 at the trimer interface that had strikingly little impact on overall trimer conformation, but critically enhanced trimer stability and improved Env antigenicity. Cross-links were also observed within gp120 at sites associated with the N241/N289 glycan hole that locally modified trimer antigenicity. In immunogenicity studies, the neutralizing antibody response to cross-linked trimers showed modest but significantly greater breadth against a global panel of difficult-to-neutralize Tier-2 heterologous viruses. Moreover, the specificity of autologous Tier-2 neutralization was modified away from the N241/N289 glycan hole, implying a novel specificity. Finally, we have investigated for the first time T helper cell responses to next-generation soluble trimers, and report on vaccine-relevant immunodominant responses to epitopes within BG505 that are modified by cross-linking. Elucidation of the structural correlates of a cross-linked viral glycoprotein will allow more rational use of this methodology for vaccine design, and reveals a strategy with promise for eliciting neutralizing antibodies needed for an effective HIV-1 vaccine.


Assuntos
HIV-1/química , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Reagentes para Ligações Cruzadas , Microscopia Crioeletrônica , Anticorpos Anti-HIV/imunologia , Antígenos HIV/química , Antígenos HIV/imunologia , Antígenos HIV/ultraestrutura , Interações Hospedeiro-Patógeno/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Coelhos , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/ultraestrutura
19.
Chemistry ; 24(38): 9485-9489, 2018 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-29653024

RESUMO

Mercaptobenzamide thioester SAMT-247 is a non-toxic, mutation-resistant HIV-1 maturation inhibitor with a unique mechanism of antiviral activity. NMR spectroscopic analyses of model reactions that mimic the cellular environment answered fundamental questions about the antiviral mechanism and inspired a high-yielding (64 % overall), scalable (75 mmol), and cost-effective ($4 mmol-1 ) three-step synthesis that will enable additional preclinical evaluation.


Assuntos
HIV-1/efeitos dos fármacos , Proteínas do Nucleocapsídeo/metabolismo , Compostos de Sulfidrila/farmacologia , HIV-1/química , Humanos , Proteínas do Nucleocapsídeo/química , Compostos de Sulfidrila/química
20.
J Virol ; 92(14)2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29618644

RESUMO

Understanding the mechanisms used by HIV-1 to evade antibody neutralization may contribute to the design of a high-coverage vaccine. The tier 3 virus 253-11 is poorly neutralized by subtype-matched and subtype C sera, even compared to other tier 3 viruses, and is also recognized poorly by V3/glycan-targeting monoclonal antibodies (MAbs). We found that sequence polymorphisms in the V3 loop and N-linked glycosylation sites contribute only minimally to the high neutralization resistance of 253-11. Interestingly, the 253-11 membrane-proximal external region (MPER) is rarely recognized by sera in the context of the wild-type virus but is commonly recognized in the context of an HIV-2 chimera, suggesting steric or kinetic hindrance of binding to MPER in the native envelope (Env). Mutations in the 253-11 MPER, which were previously reported to increase the lifetime of the prefusion Env conformation, affected the resistance of 253-11 to antibodies targeting various epitopes on HIV-1 Env, presumably destabilizing its otherwise stable, closed trimer structure. To gain insight into the structure of 253-11, we constructed and crystallized a recombinant 253-11 SOSIP trimer. The resulting structure revealed that the heptad repeat helices in gp41 are drawn in close proximity to the trimer axis and that gp120 protomers also showed a relatively compact disposition around the trimer axis. These observations give substantial insight into the molecular features of an envelope spike from a tier 3 virus and into possible mechanisms that may contribute to its unusually high neutralization resistance.IMPORTANCE HIV-1 isolates that are highly resistant to broadly neutralizing antibodies could limit the efficacy of an antibody-based vaccine. We studied 253-11, which is highly resistant to commonly elicited neutralizing antibodies. To further understand its resistance, we made mutations that are known to delay fusion and thus increase the time that the virus spends in the open conformation following CD4 binding. Interestingly, we found that these mutations affect the 253-11 envelope (Env) spike before CD4 binding, presumably by destabilizing the trimer structure. To gain further information about the structure of the 253-11 Env trimer, we generated a recombinant 253-11 SOSIP trimer. The crystal structure of the SOSIP trimer revealed that the gp41 helices and the gp120 protomers were drawn in toward the center of the molecule compared to most solved HIV-1 Env structures. These observations provide insight into the distinct molecular features of a tier 3 envelope spike.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/genética , HIV-1/imunologia , Sequência de Aminoácidos , Epitopos/imunologia , Proteína gp120 do Envelope de HIV/química , Infecções por HIV/virologia , HIV-1/química , Humanos , Polimorfismo Genético , Polissacarídeos/imunologia
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