RESUMO
The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1-8. Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)8-10. It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers)5,11-18. Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1JR-FL strain in complex with the antibody PGT15119. Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.
Assuntos
Transferência Ressonante de Energia de Fluorescência , HIV-1/química , Imagem Individual de Molécula , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Animais , Anticorpos Neutralizantes/imunologia , Bovinos , Dissulfetos/química , Células HEK293 , HIV-1/genética , HIV-1/imunologia , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer of gp120-gp41 heterodimers mediates virus entry into CD4-positive (CD4+) cells. Single-molecule fluorescence resonance energy transfer (smFRET) has revealed that native Env on the surface of viruses predominantly exists in a pretriggered conformation (state 1) that is preferentially recognized by many broadly neutralizing antibodies (bNAbs). Env is activated by binding receptor CD4, which drives transitions through a default intermediate conformation (state 2) into the three-CD4-bound open conformation (state 3). The application of smFRET to assess the conformational state of existing Env constructs and ligand complexes recently revealed that all current high-resolution structures correspond to downstream states 2 and 3. The structure of state 1, therefore, remains unknown. We sought to identify conditions whereby HIV-1 Env could be stabilized in the pretriggered state 1 for possible structural characterization. Shedding of gp120, known to severely complicate structural studies, can be prevented by using the uncleaved gp160JR-FL precursor with alterations in the protease cleavage site (R508S/R511S) or by introducing a disulfide bridge between gp120 and gp41 designated "SOS" (A501C/T605C). smFRET demonstrated that both shedding-preventing modifications shifted the conformational landscape of Env downstream toward states 2 and 3. However, both membrane-bound Env proteins on the surface of intact viruses remained conformationally dynamic, responsive to state-stabilizing ligands, and able to be stabilized in state 1 by specific ligands such as the Bristol-Myers Squibb (BMS) entry inhibitors. The here-described identification of state 1-stabilizing conditions may enable structural characterization of the state 1 conformation of HIV-1 Env.IMPORTANCE The HIV-1 envelope glycoprotein (Env) opens in response to receptor CD4 binding from a pretriggered (state 1) conformation through a necessary intermediate to the three-CD4-bound conformation. The application of smFRET to test the conformational state of existing Env constructs and ligand complexes used for high-resolution structures recently revealed that they correspond to the downstream conformations. The structure of the pretriggered Env conformation, preferentially recognized by broadly neutralizing antibodies, remains unknown. Here, we identify experimental conditions that stabilize membrane-bound and shedding-resistant virus Env trimers in state 1, potentially facilitating structural characterization of this unknown conformational state.
Assuntos
Glicoproteínas/química , Glicoproteínas/imunologia , HIV-1/imunologia , Eliminação de Partículas Virais/imunologia , Eliminação de Partículas Virais/fisiologia , Anticorpos Neutralizantes/imunologia , Antígenos CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Dissulfetos , Células HEK293 , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/imunologia , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Internalização do Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Quorum sensing (QS) signaling regulates the motility, adhesion, and biofilm formation of bacteria, and at the same time activates immune response in eukaryotic organisms. We recently demonstrated that the QS molecule, dihydroxy-2, 3-pentanedione (DPD), and its analogs significantly inhibit estradiol-regulated virulence of Prevotella aurantiaca, one of the four species in the Prevotella intermedia group. Here, we examined the combined effects of estradiol and QS signaling on 1) cytokine response of human gingival keratinocytes (HMK) against whole cell extract (WCE) of P. intermedia, Prevotella nigrescens, and Prevotella pallens, and 2) biofilm formation of these three Prevotella species. All experiments were performed in the presence or absence of estradiol, and with different QS molecules: DPD and its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD). Concentrations of interleukin (IL)-1ß, -6, and -8 were determined by the Luminex multiplex immunoassay, biofilm mass was quantitatively evaluated by measuring protein concentration via the Bradford method, and the microtopography of biofilms was assessed by scanning electron microscopy (SEM) imaging. Concentrations of IL-6 and IL-8 were elevated when HMK cells were incubated with estradiol and WCE of P. intermedia and P. nigrescens, but decreased when incubated with estradiol and WCE of P. pallens. Butyl-DPD neutralized the estradiol- and WCE-induced regulation of HMK interleukin expression and, at the same time, inhibited the biofilm formation of P. intermedia and P. nigrescens. SEM micrographs revealed a decrease in biofilm mass after application of butyl-DPD, which was most detectable among the P. intermedia ATCC 25611 and P. nigrescens ATCC 33563 and AHN 8293 strains. In conclusion, butyl-DPD analog is able to neutralize the WCE-induced epithelial cytokine response and, at the same time, to inhibit the biofilm formation of P. intermedia and P. nigrescens.
Assuntos
Infecções por Bacteroidaceae/imunologia , Células Epiteliais/imunologia , Gengiva/imunologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Interleucina-8/imunologia , Prevotella/fisiologia , Percepção de Quorum , Infecções por Bacteroidaceae/genética , Infecções por Bacteroidaceae/microbiologia , Biofilmes , Células Epiteliais/microbiologia , Gengiva/microbiologia , Humanos , Interleucina-1beta/genética , Interleucina-6/genética , Interleucina-8/genética , Queratinócitos/imunologia , Queratinócitos/microbiologia , Prevotella/classificação , Prevotella/genética , Prevotella/patogenicidade , Prevotella intermedia/genética , Prevotella intermedia/patogenicidade , Prevotella intermedia/fisiologia , Prevotella nigrescens/genética , Prevotella nigrescens/patogenicidade , Prevotella nigrescens/fisiologiaRESUMO
Biofilm formation and dipeptidyl peptidase IV (DPPIV) enzyme activity contribute to the virulence of oral bacteria, and these virulence factors are partly regulated by quorum sensing signaling system. We recently demonstrated that estradiol regulates growth properties and DPPIV activity of Prevotella intermedia, Prevotella nigrescens, and Prevotella pallens. Here, we examined the DPPIV dependency of biofilm formation of Prevotella aurantiaca. Three strains (two clinical strains AHN 37505 and 37552 and the type strain CCUG 57723) were incubated in three estradiol concentrations (30, 90, and 120 nmol/L). Regulation of DPPIV activity, biofilm and fimbria formation, and coaggregation of bacterial strains were analyzed after incubation with four concentrations (10 nM, 100 nM, 1 µM, 10 µM) of dihydroxy-2,3-pentaedione (DPD), the universal precursor of autoinducer -2 (AI-2), and analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD) for 24 h. Estradiol enhanced the planktonic growth, coaggregation, and biofilm formation of P. aurantiaca strains. The whole cell extract of AHN 37505 had the highest DPPIV activity, followed by CCUG 57723 and AHN 37552. Inhibition of DPPIV activity with di-isopropylfluorophosphate suppressed the effect of estradiol on biofilm formation. At 100 nM and 10 µM concentrations of DPD, butyl DPD, and isobutyl DPD, biofilm formation of P. aurantiaca was significantly inhibited. Fimbriae formation was enhanced up to concentrations of 100 nM and 1 µM followed by a significant inhibition at higher concentrations of DPD and all analogs. A slight but significant inhibitory effect of DPD and analogs on DPPIV activity was observed. Our results indicate that DPPIV plays a key role in the estradiol-regulated biofilm formation of P. aurantiaca. Quorum sensing autoinducer DPD and C1-alkyl analogs could inhibit biofilm-related virulence of P. aurantiaca.
Assuntos
Infecções por Bacteroidaceae/microbiologia , Biofilmes/crescimento & desenvolvimento , Dipeptidil Peptidase 4/metabolismo , Prevotella/fisiologia , Percepção de Quorum , Transdução de Sinais , Biofilmes/efeitos dos fármacos , Ativação Enzimática , Estradiol/farmacologia , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Prevotella/patogenicidade , Prevotella/ultraestrutura , Virulência , Fatores de VirulênciaRESUMO
An elegant synergistic catalytic system comprising a ruthenium complex with a chiral Brønsted acid was developed for a four-component Mannich/cascade aza-Michael reaction. The ruthenium-associated ammonium ylides successfully trapped with in situ generated imines indicates a stepwise process of proton transfer in the ruthenium-catalyzed carbenoid N-H insertion reaction. The different decomposition abilities of various ruthenium complexes towards diazo compounds were well explained by the calculated thermodynamic data. The transformation features a mild, rapid, and efficient method for the synthesis of enantiomerically pure 1,3,4-tetrasubstituted tetrahydroquinolines bearing a quaternary stereogenic carbon center from simple starting precursors in moderate yields with high diastereo- and enantioselectivity.
Assuntos
Rutênio/química , Tetra-Hidroisoquinolinas/síntese química , Catálise , Complexos de Coordenação/química , Estereoisomerismo , Tetra-Hidroisoquinolinas/químicaRESUMO
The chiral Rh(I)-diene-catalyzed asymmetric three-component reaction of aryldiazoacetates, aromatic amines, and ß-nitroacrylates was achieved to obtain γ-nitro-α-amino-succinates in good yields and with high diastereo- and enantioselectivity. This reaction is proposed to proceed through the enantioselective trapping of Rh(I)-associated ammonium ylides by nitroacrylates. This new transformation represents the first example of Rh(I)-carbene-induced ylide transformation.
Assuntos
Acrilatos/química , Compostos de Amônio/química , Metano/análogos & derivados , Ródio/química , Catálise , Metano/química , Estrutura Molecular , EstereoisomerismoRESUMO
A xenotropic murine leukemia virus-related virus (XMRV) has been reported to be an emerging pathogen associated with prostate cancer (PC) and chronic fatigue syndrome (CFS). However, recent studies have demonstrated that XMRV is a laboratory-derived virus resulting from genetic recombination between two mouse viral genomes during serial xenograft tissue transplantation. This study describes a phylogenetic analysis that compared XMRV with the ecotropic murine leukemia viruses (E-MLV), xenotropic MLV (X-MLV), and other retroviruses, including HTLV-1 and HIV-1. We found that sequences corresponding to three XMRV structural proteins (Env, Gag, and Pol) exhibited high degrees of homology with X-MLV (>91 %) and E-MLV (67-96 %), but not HTLV-1 (13-16 %) or HIV-1 (10-15 %), indicating that XMRV was derived from X-MLV and/or E-MLV. We then compared the infectivity of XMRV and E-MLV for human and murine lymphocytes, respectively. Results showed that human PBMCs were not susceptible to XMRV infection, suggesting that XMRV exhibits host cell tropism similar to E-MLV that only infects murine PBMCs. These data suggest that it is unlikely that this laboratory-generated retrovirus could cause disease in humans.
Assuntos
DNA Viral/genética , Filogenia , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/classificação , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/patogenicidade , Animais , Análise por Conglomerados , Produtos do Gene env/genética , Produtos do Gene gag/genética , Produtos do Gene pol/genética , Humanos , Leucócitos/virologia , Camundongos , Homologia de Sequência de Aminoácidos , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/genéticaRESUMO
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/metabolismoRESUMO
The continuing emergence and spread of antibiotic-resistant bacteria is worrisome and new strategies to curb bacterial infections are being sought. The interference of bacterial quorum sensing (QS) signaling has been suggested as a prospective antivirulence strategy. The AI-2 QS system is present in multiple bacterial species and has been shown to be correlated with pathogenicity. To facilitate the discovery of novel compounds interfering with AI-2 QS, we established a high-throughput setup of whole-cell bioreporter assay, which can be performed in either 96- or 384-well format. Agonistic or antagonistic activities of the test compounds against Escherichia coli LsrB-type AI-2 QS system are monitored by measuring the level of ß-galactosidase expression. A control strain expressing ß-galactosidase in quorum sensing-independent manner is included into the assay for false-positive detection.
Assuntos
Bactérias/metabolismo , Bioensaio/métodos , Descoberta de Drogas/métodos , Percepção de Quorum , Proteínas Arqueais/genética , Bactérias/efeitos dos fármacos , Bactérias/genética , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Pentanonas/antagonistas & inibidores , Transdução de Sinais , beta-Galactosidase/metabolismoRESUMO
Quorum sensing (QS), a bacterial process that regulates population-scale behavior, is mediated by small signaling molecules, called autoinducers (AIs), that are secreted and perceived, modulating a "collective" phenotype. Because the autoinducer AI-2 is secreted by a wide variety of bacterial species, its "perception" cues bacterial behavior. This response is mediated by the lsr (LuxS-regulated) operon that includes the AI-2 transporter LsrACDB and the kinase LsrK. We report that HPr, a phosphocarrier protein central to the sugar phosphotransferase system of Escherichia coli, copurifies with LsrK. Cocrystal structures of an LsrK/HPr complex were determined, and the effects of HPr and phosphorylated HPr on LsrK activity were assessed. LsrK activity is inhibited when bound to HPr, revealing new linkages between QS activity and sugar metabolism. These findings help shed new light on the abilities of bacteria to rapidly respond to changing nutrient levels at the population scale. They also suggest new means of manipulating QS activity among bacteria and within various niches.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Percepção de Quorum , Açúcares/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Metabolismo dos Carboidratos , Ativação Enzimática , Proteínas de Escherichia coli/química , Regulação Bacteriana da Expressão Gênica , Cinética , Modelos Moleculares , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
The original version of this Article contained an error in the spelling of the author Amos B. Smith, III, which was incorrectly given as Amos B. SmithIII. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMO
HIV-1 entry into cells requires binding of the viral envelope glycoprotein (Env) to receptor CD4 and coreceptor. Imaging of individual Env molecules on native virions shows Env trimers to be dynamic, spontaneously transitioning between three distinct well-populated conformational states: a pre-triggered Env (State 1), a default intermediate (State 2) and a three-CD4-bound conformation (State 3), which can be stabilized by binding of CD4 and coreceptor-surrogate antibody 17b. Here, using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we show the default intermediate configuration to be asymmetric, with individual protomers adopting distinct conformations. During entry, this asymmetric intermediate forms when a single CD4 molecule engages the trimer. The trimer can then transition to State 3 by binding additional CD4 molecules and coreceptor.
Assuntos
HIV-1/metabolismo , Conformação Proteica , Multimerização Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Antígenos CD4/química , Antígenos CD4/metabolismo , Transferência Ressonante de Energia de Fluorescência , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Vírion/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Acute myeloid leukemia (AML) is an aggressive malignancy with only a handful of therapeutic options. About 30% of AML patients harbor mutated FLT3 kinase, and thus, this cancer-driver has become a hotly pursued AML target. Herein we report a new class of FLT3 inhibitors, which potently inhibit the proliferation of acute myeloid leukemia (AML) cells at nanomolar concentrations.
RESUMO
AIM: Mutated or overexpressed FLT3 drives about 30% of reported acute myeloid leukemia (AML). Currently, FLT3 inhibitors have shown durable clinical responses but a complete remission of AML with FLT3 inhibitors remains elusive due to mutation-driven resistance mechanisms. The development of FLT3 inhibitors that also target other downstream oncogenic kinases may combat the resistance mechanism. RESULTS: 4-substituted aminoisoquinoline benzamides potently inhibit Src-family kinases and FLT3, including secondary mutations, such as FLT3D835. Modifications of aminoisoquinoline benzamide to aminoquinoline or aminoquinazoline abrogated FLT3 and Src-family kinase binding. CONCLUSION: The lead aminoisoquinolines potently inhibited FLT3-driven AML cell lines, MV4-11 and MOLM-14. These aminoisoquinoline benzamides represent new kinase scaffolds with high potential to be translated into anticancer agents.
Assuntos
Antineoplásicos/síntese química , Benzamidas/síntese química , Isoquinolinas/síntese química , Leucemia Mieloide Aguda/tratamento farmacológico , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Quinases da Família src/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose , Benzamidas/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Humanos , Isoquinolinas/farmacologia , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Conformação ProteicaRESUMO
The entry of HIV-1 into target cells is mediated by the viral envelope glycoproteins (Env). Binding to the CD4 receptor triggers a cascade of conformational changes in distant domains that move Env from a functionally "closed" State 1 to more "open" conformations, but the molecular mechanisms underlying allosteric regulation of these transitions are still elusive. Here, we develop chemical probes that block CD4-induced conformational changes in Env and use them to identify a potential control switch for Env structural rearrangements. We identify the gp120 ß20-ß21 element as a major regulator of Env transitions. Several amino acid changes in the ß20-ß21 base lead to open Env conformations, recapitulating the structural changes induced by CD4 binding. These HIV-1 mutants require less CD4 to infect cells and are relatively resistant to State 1-preferring broadly neutralizing antibodies. These data provide insights into the molecular mechanism and vulnerability of HIV-1 entry.
Assuntos
Proteína gp120 do Envelope de HIV/química , Antígenos CD4/metabolismo , Células HEK293 , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Humanos , Sondas Moleculares , Ligação Proteica , Conformação Proteica , Conformação Proteica em Folha betaRESUMO
Primary human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimers [(gp120/gp41)3] typically exist in a metastable closed conformation (state 1). Binding the CD4 receptor triggers Env to undergo extensive conformational changes to mediate virus entry. We identified specific gp120 residues that restrain Env in state 1. Alteration of these restraining residues destabilized state 1, allowing Env to populate a functional conformation (state 2) intermediate between state 1 and the full CD4-bound state (state 3). Increased state 2 occupancy was associated with lower energy barriers between the states. State 2 was an obligate intermediate for all transitions between state 1 and state 3. State 2-enriched Envs required lower CD4 concentrations to trigger virus entry and more efficiently infected cells expressing low levels of CD4. These Envs were resistant to several broadly neutralizing antibodies and small-molecule inhibitors. Thus, state 2 is an Env conformation on the virus entry pathway; sampling state 2 increases the adaptability of HIV-1 to different host cell receptor levels and immune environments. Our results provide new insights into the conformational regulation of HIV-1 entry. IMPORTANCE: The envelope glycoproteins (Env) of HIV-1 mediate virus entry and are the sole targets of neutralizing antibodies. Understanding the way that Env promotes HIV-1 entry can expedite drug and vaccine development. By destabilizing Env, we found that it assumes an intermediate state that is functional and obligate for transitions to entry-competent conformations. Increased sampling of this state enhances the ability of HIV-1 to infect cells that express low levels of the CD4 receptor and allows the virus to evade neutralizing antibodies and small-molecule inhibitors. These findings provide new mechanistic insights into the function and inhibition of HIV-1 Env and will contribute to ongoing therapeutic and prevention efforts to combat HIV-1.
Assuntos
Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , HIV-1/fisiologia , Proteínas Mutantes/química , Proteínas Mutantes/genética , Internalização do Vírus , Antígenos CD4/metabolismo , Linhagem Celular , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação ProteicaRESUMO
The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. Here, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showed that the N-terminal portion of the fusion peptide can be solvent-exposed. These results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Proteínas Virais de Fusão/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/ultraestrutura , Linfócitos B/imunologia , Linfócitos B/virologia , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Epitopos Imunodominantes/imunologia , Dados de Sequência Molecular , Peptídeos/imunologia , Conformação Proteica , Internalização do VírusRESUMO
As the sole viral antigen on the HIV-1-virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1-Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.