RESUMEN
HIV-1 envelope glycoprotein (Env), which consists of trimeric (gp160)3 cleaved to (gp120 and gp41)3, interacts with the primary receptor CD4 and a coreceptor (such as chemokine receptor CCR5) to fuse viral and target-cell membranes. The gp120-coreceptor interaction has previously been proposed as the most crucial trigger for unleashing the fusogenic potential of gp41. Here we report a cryo-electron microscopy structure of a full-length gp120 in complex with soluble CD4 and unmodified human CCR5, at 3.9 Å resolution. The V3 loop of gp120 inserts into the chemokine-binding pocket formed by seven transmembrane helices of CCR5, and the N terminus of CCR5 contacts the CD4-induced bridging sheet of gp120. CCR5 induces no obvious allosteric changes in gp120 that can propagate to gp41; it does bring the Env trimer close to the target membrane. The N terminus of gp120, which is gripped by gp41 in the pre-fusion or CD4-bound Env, flips back in the CCR5-bound conformation and may irreversibly destabilize gp41 to initiate fusion. The coreceptor probably functions by stabilizing and anchoring the CD4-induced conformation of Env near the cell membrane. These results advance our understanding of HIV-1 entry into host cells and may guide the development of vaccines and therapeutic agents.
Asunto(s)
Antígenos CD4/química , Antígenos CD4/ultraestructura , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/ultraestructura , Receptores CCR5/química , Receptores CCR5/ultraestructura , Receptores del VIH/química , Receptores del VIH/ultraestructura , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Sitios de Unión , Antígenos CD4/aislamiento & purificación , Antígenos CD4/metabolismo , Línea Celular , Quimiocina CCL5/química , Quimiocina CCL5/metabolismo , Proteína gp120 de Envoltorio del VIH/aislamiento & purificación , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/ultraestructura , Humanos , Ligandos , Maraviroc/química , Maraviroc/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores CCR5/aislamiento & purificación , Receptores CCR5/metabolismo , Receptores del VIH/antagonistas & inhibidores , Receptores del VIH/metabolismoRESUMEN
Because of the level of attention it received due to its role as the principal HIV coreceptor, CCR5 has been described as a 'celebrity' chemokine receptor. Here we describe the development of CCR5 inhibitory strategies that have been developed for HIV therapy and which are now additionally being considered for use in HIV prevention and cure. The wealth of CCR5-related tools that have been developed during the intensive investigation of CCR5 as an HIV drug target can now be turned towards the study of CCR5 as a model chemokine receptor. We also summarize what is currently known about the cell biology and pharmacology of CCR5, providing an update on new areas of investigation that have emerged in recent research. Finally, we discuss the potential of CCR5 as a drug target for diseases other than HIV, discussing the evidence linking CCR5 and its natural chemokine ligands with inflammatory diseases, particularly neuroinflammation, and certain cancers. These pathologies may provide new uses for the strategies for CCR5 blockade originally developed to combat HIV/AIDS.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/prevención & control , VIH-1/metabolismo , Receptores CCR5/metabolismo , Receptores del VIH/antagonistas & inhibidores , Receptores del VIH/metabolismo , Humanos , Inflamación/patología , Transducción de Señal/fisiologíaRESUMEN
UNLABELLED: Gene-engineered CD34(+) hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34(+) cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4(+) T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIV in vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34(+) cells that produce a constant supply of HIV-resistant progeny. IMPORTANCE: Major issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4(+) T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may be beneficial not only for HIV patients but also for other patients requiring transplantation of genetically modified cells.
Asunto(s)
Resistencia a la Enfermedad , Técnicas de Silenciamiento del Gen , Infecciones por VIH/inmunología , VIH-1/fisiología , Receptores CCR5/metabolismo , Receptores del VIH/antagonistas & inhibidores , Acoplamiento Viral , Animales , Terapia Genética/métodos , Vectores Genéticos , Infecciones por VIH/virología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/virología , Humanos , Lentivirus/genética , Ratones SCID , Trasplante , Carga ViralRESUMEN
OBJECTIVES: This study measured and compared the pharmacokinetics of CMPD167, a small molecule antiretroviral CCR5 inhibitor with potential as an HIV microbicide, following vaginal, rectal and oral administration in rhesus macaques. METHODS: A vaginal hydroxyethylcellulose (HEC) gel, a rectal HEC gel, a silicone elastomer matrix-type vaginal ring and an oral solution, each containing CMPD167, were prepared and administered to rhesus macaques pretreated with Depo-Provera. CMPD167 concentrations in vaginal fluid, vaginal tissue (ring only), rectal fluid and blood plasma were quantified by HPLC-mass spectrometry. RESULTS: CMPD167 concentrations measured in rectal fluid, vaginal fluid and blood plasma were highly dependent on both the route of administration and the formulation type. Although rectal and vaginal fluid concentrations were highest when CMPD167 was administered locally (via either gel or ring), lower concentrations of the drug were also measured in these compartments following administration at the remote mucosal site or orally. CMPD167 levels in the vaginal and rectal fluid following oral administration were relatively low compared with local administration. CONCLUSIONS: The study provides clear evidence for vaginal-rectal and rectal-vaginal drug transfer pathways and suggests that oral pre-exposure prophylaxis with CMPD167 may be less efficacious at preventing sexual transmission of HIV-1 than topically applied products.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacocinética , Antagonistas de los Receptores CCR5/administración & dosificación , Antagonistas de los Receptores CCR5/farmacocinética , Pirazoles/administración & dosificación , Pirazoles/farmacocinética , Receptores del VIH/antagonistas & inhibidores , Valina/análogos & derivados , Administración Intravaginal , Administración Oral , Administración Rectal , Animales , Líquidos Corporales/química , Cromatografía Líquida de Alta Presión , Femenino , Macaca mulatta , Masculino , Espectrometría de Masas , Valina/administración & dosificación , Valina/farmacocinéticaRESUMEN
The membrane-bound chemokine fractalkine (FKN, CX3CL1) on endothelial cells plays a role in leukocyte trafficking. The chemokine domain (FKN-CD) is sufficient for inducing FKN signaling (e.g., integrin activation), and FKN-CD binds to its receptor CX3CR1 on leukocytes. Whereas previous studies suggest that FKN-CD does not directly bind to integrins, our docking simulation studies predicted that FKN-CD directly interacts with integrin α(v)ß(3). Consistent with this prediction, we demonstrated that FKN-CD directly bound to α(v)ß(3) and α(4)ß(1) at a very high affinity (K(D) of 3.0 × 10(-10) M to α(v)ß(3) in 1 mM Mn(2+)). Also, membrane-bound FKN bound to integrins α(v)ß(3) and α(4)ß(1), suggesting that the FKN-CD/integrin interaction is biologically relevant. The binding site for FKN-CD in α(v)ß(3) was similar to those for other known α(v)ß(3) ligands. Wild-type FKN-CD induced coprecipitation of integrins and CX3CR1 in U937 cells, suggesting that FKN-CD induces ternary complex formation (CX3CR1, FKN-CD, and integrin). Based on the docking model, we generated an integrin-binding defective FKN-CD mutant (the K36E/R37E mutant). K36E/R37E was defective in ternary complex formation and integrin activation, whereas K36E/R37E still bound to CX3CR1. These results suggest that FKN-CD binding to CX3CR1 is not sufficient for FKN signaling, and that FKN-CD binding to integrins as coreceptors and the resulting ternary complex formation are required for FKN signaling. Notably, excess K36E/R37E suppressed integrin activation induced by wild-type FKN-CD and effectively suppressed leukocyte infiltration in thioglycollate-induced peritonitis. These findings suggest that K36E/R37E acts as a dominant-negative CX3CR1 antagonist and that FKN-CD/integrin interaction is a novel therapeutic target in inflammatory diseases.
Asunto(s)
Quimiocina CX3CL1/genética , Quimiocina CX3CL1/metabolismo , Integrina alfa4beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Receptores de Citocinas/antagonistas & inhibidores , Receptores de Citocinas/metabolismo , Receptores del VIH/antagonistas & inhibidores , Receptores del VIH/metabolismo , Animales , Células CHO , Receptor 1 de Quimiocinas CX3C , Quimiocinas/metabolismo , Simulación por Computador , Cricetinae , Cricetulus , Humanos , Células K562 , Mutación/genética , Unión Proteica/genética , Unión Proteica/inmunología , Estructura Terciaria de Proteína/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Células U937RESUMEN
BACKGROUND: HIV-1 entry into target lymphocytes requires the activity of actin adaptors that stabilize and reorganize cortical F-actin, like moesin and filamin-A. These alterations are necessary for the redistribution of CD4-CXCR4/CCR5 to one pole of the cell, a process that increases the probability of HIV-1 Envelope (Env)-CD4/co-receptor interactions and that generates the tension at the plasma membrane necessary to potentiate fusion pore formation, thereby favouring early HIV-1 infection. However, it remains unclear whether the dynamic processing of F-actin and the amount of cortical actin available during the initial virus-cell contact are required to such events. RESULTS: Here we show that gelsolin restructures cortical F-actin during HIV-1 Env-gp120-mediated signalling, without affecting cell-surface expression of receptors or viral co-receptor signalling. Remarkably, efficient HIV-1 Env-mediated membrane fusion and infection of permissive lymphocytes were impaired when gelsolin was either overexpressed or silenced, which led to a loss or gain of cortical actin, respectively. Indeed, HIV-1 Env-gp120-induced F-actin reorganization and viral receptor capping were impaired under these experimental conditions. Moreover, gelsolin knockdown promoted HIV-1 Env-gp120-mediated aberrant pseudopodia formation. These perturbed-actin events are responsible for the inhibition of early HIV-1 infection. CONCLUSIONS: For the first time we provide evidence that through its severing of cortical actin, and by controlling the amount of actin available for reorganization during HIV-1 Env-mediated viral fusion, entry and infection, gelsolin can constitute a barrier that restricts HIV-1 infection of CD4+ lymphocytes in a pre-fusion step. These findings provide important insights into the complex molecular and actin-associated dynamics events that underlie early viral infection. Thus, we propose that gelsolin is a new factor that can limit HIV-1 infection acting at a pre-fusion step, and accordingly, cell-signals that regulate gelsolin expression and/or its actin-severing activity may be crucial to combat HIV-1 infection.
Asunto(s)
Actinas/antagonistas & inhibidores , Antivirales/metabolismo , Linfocitos T CD4-Positivos/inmunología , Gelsolina/metabolismo , VIH-1/inmunología , Receptores del VIH/antagonistas & inhibidores , Internalización del Virus , Antivirales/uso terapéutico , Linfocitos T CD4-Positivos/virología , Línea Celular , VIH-1/fisiología , Humanos , Transducción de SeñalRESUMEN
CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454 sequencing using Geno2Pheno at a 10% FPR and 0.3% threshold and VERITROP more accurately predicted the success of a maraviroc-based regimen. In conclusion, VERITROP may promote the development of new HIV coreceptor antagonists and aid in the treatment and management of HIV-infected individuals prior to and/or during treatment with this class of drugs.
Asunto(s)
VIH-1/fisiología , Receptores CCR5/genética , Receptores CXCR4/genética , Receptores del VIH/antagonistas & inhibidores , Receptores del VIH/metabolismo , Tropismo Viral , Fármacos Anti-VIH/farmacología , Fusión Celular , Línea Celular , Ciclohexanos , Genoma Viral , Genotipo , Células HEK293 , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Humanos , Maraviroc , Proyectos Piloto , ARN Viral/genética , Proteínas de Saccharomyces cerevisiae/genética , Sensibilidad y Especificidad , Triazoles , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
MOTIVATION: Due to the high mutation rate of human immunodeficiency virus (HIV), drug-resistant-variants emerge frequently. Therefore, researchers are constantly searching for new ways to attack the virus. One new class of anti-HIV drugs is the class of coreceptor antagonists that block cell entry by occupying a coreceptor on CD4 cells. This type of drug just has an effect on the subset of HIVs that use the inhibited coreceptor. A good prediction of whether the viral population inside a patient is susceptible to the treatment is hence very important for therapy decisions and pre-requisite to administering the respective drug. The first prediction models were based on data from Sanger sequencing of the V3 loop of HIV. Recently, a method based on next-generation sequencing (NGS) data was introduced that predicts labels for each read separately and decides on the patient label through a percentage threshold for the resistant viral minority. RESULTS: We model the prediction problem on the patient level taking the information of all reads from NGS data jointly into account. This enables us to improve prediction performance for NGS data, but we can also use the trained model to improve predictions based on Sanger sequencing data. Therefore, also laboratories without NGS capabilities can benefit from the improvements. Furthermore, we show which amino acids at which position are important for prediction success, giving clues on how the interaction mechanism between the V3 loop and the particular coreceptors might be influenced. AVAILABILITY: A webserver is available at http://coreceptor.bioinf.mpi-inf.mpg.de. CONTACT: nico.pfeifer@mpi-inf.mpg.de.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Secuenciación de Nucleótidos de Alto Rendimiento , Receptores del VIH/antagonistas & inhibidores , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Humanos , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Carga Viral , Internalización del Virus/efectos de los fármacos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The membrane-anchored CX3C chemokine fractalkine (FKN) is expressed on activated endothelium and is associated with the development of atherosclerosis. The potential of FKN in mediating platelet adhesion beyond platelet activation remains unexplored to date. A flow-based adhesion assay was used to study the adhesion of platelets to immobilized FKN under physiologic flow conditions. Platelet adhesion to von Willebrand factor (VWF) was increased in the presence of FKN at 600 inverse seconds. Additional platelet adhesion to FKN coimmobilized with VWF was dependent on the FKN receptor CX3CR1 and activation of glycoprotein (GP) IIb/IIIa. The number of platelets rolling on VWF was likewise enhanced in the presence of FKN. The enhancement of rolling on FKN and VWF was insensitive to anti-CX3CR1 antibody but was fully inhibited by neutralizing GPIbα function. The extracellular domain of GPIbα was covalently coupled to fluorescent microspheres, and microsphere binding was significantly higher in the presence of FKN. Platelet adhesion to activated endothelium in vitro and to intact human arteries was substantially increased in an FKN-dependent manner. These data demonstrate that endothelial expressed FKN activates platelets via its cognate receptor CX3CR1, whereas platelet adhesion is predominantly mediated by GPIbα and independent of CX3CR1.
Asunto(s)
Quimiocina CX3CL1/fisiología , Adhesividad Plaquetaria/fisiología , Complejo GPIb-IX de Glicoproteína Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiología , Factor de von Willebrand/fisiología , Arterias/fisiología , Receptor 1 de Quimiocinas CX3C , Células Endoteliales/fisiología , Hemorreología , Humanos , Técnicas In Vitro , Complejo GPIb-IX de Glicoproteína Plaquetaria/antagonistas & inhibidores , Receptores de Citocinas/antagonistas & inhibidores , Receptores de Citocinas/fisiología , Receptores del VIH/antagonistas & inhibidores , Receptores del VIH/fisiologíaRESUMEN
We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patient's pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4(+) T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N' terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a tropism shift toward effector memory cells upon infection of primary CD4(+) T cells in the presence of APL, with relative sparing of the central memory CD4(+) T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses, then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T(CM) subset of CD4(+) T cells and result in improved T cell homeostasis and immune function.
Asunto(s)
Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/virología , Farmacorresistencia Viral , VIH-1/efectos de los fármacos , Receptores CCR5/fisiología , Receptores del VIH/fisiología , Tropismo Viral , Benzoatos/farmacología , Antagonistas de los Receptores CCR5 , Dicetopiperazinas , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/fisiología , Humanos , Mutación Missense , Piperazinas/farmacología , Receptores del VIH/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Acoplamiento ViralRESUMEN
CXC-chemokine receptor (CXCR)-4/fusin, a newly discovered co-receptor for T-cell line (T)-tropic HIV-1 virus, plays a critical role in T-tropic virus fusion and entry into permissive cells. The occurrence of T-tropic HIV viruses is associated with CD4-positive cell decline and progression to AIDS, suggesting that the T-tropic HIV-1 contributes to AIDS pathogenesis. In this study, we used a novel strategy to inactivate CXCR-4 by targeting a modified CXC-chemokine to the endoplasmic reticulum (ER) to block the surface expression of newly synthesized CXCR-4. The genetically modified lymphocytes expressing this intracellular chemokine, termed "intrakine", are immune to T-tropic virus infection and appear to retain normal biological features. Thus, this genetic intrakine strategy is uniquely targeted at the conserved cellular receptor for the prevention of HIV-1 entry and may be developed into an effective treatment for HIV-1 infection and AIDS.
Asunto(s)
Quimiocinas CXC , Quimiocinas/biosíntesis , VIH-1/fisiología , Linfocitos/inmunología , Receptores CXCR4/antagonistas & inhibidores , Receptores del VIH/antagonistas & inhibidores , Síndrome de Inmunodeficiencia Adquirida/terapia , Linfocitos B/inmunología , Línea Celular , Línea Celular Transformada , Células Cultivadas , Quimiocina CXCL12 , Citomegalovirus , Vectores Genéticos , Células Gigantes , Células HeLa , Humanos , Reacción en Cadena de la Polimerasa , Receptores CXCR4/biosíntesis , Receptores CXCR4/inmunología , Receptores del VIH/inmunología , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Transfección , Células Tumorales CultivadasRESUMEN
Identification of cellular factors involved in HIV-1 entry and transmission at mucosal surfaces is critical for understanding viral pathogenesis and development of effective prevention strategies. Here we describe the evaluation of HIV-1 entry inhibitors for their ability to prevent infection of, and dissemination from, human cervical tissue ex vivo. Blockade of CD4 alone or CCR5 and CXCR4 together inhibited localized mucosal infection. However, simultaneous blockade of CD4 and mannose-binding C-type lectin receptors including dendritic cell-specific intercellular adhesion molecule-grabbing integrin was required to inhibit HIV-1 uptake and dissemination by migratory cells. In contrast, direct targeting of HIV-1 by neutralizing mAb b12 and CD4-IgG2 (PRO-542) blocked both localized infection and viral dissemination pathways. Flow cytometric analysis and immunostaining of migratory cells revealed two major populations, CD3(+)HLA-DR(-) and CD3(-)HLA-DR(+) cells, with a significant proportion of the latter also expressing dendritic cell-specific intercellular adhesion molecule-grabbing integrin. Bead depletion studies demonstrated that such HLA-DR(+) cells accounted for as much as 90% of HIV-1 dissemination. Additional studies using immature monocyte-derived dendritic cells demonstrated that although mannose-binding C-type lectin receptors and CD4 are the principal receptors for gp120, other mechanisms may account for virus capture. Our identification of the predominant receptors involved in HIV-1 infection and dissemination within human cervical tissue highlight important targets for microbicide development.
Asunto(s)
Cuello del Útero/virología , Infecciones por VIH/prevención & control , Receptores del VIH/antagonistas & inhibidores , Antagonistas de los Receptores CCR5 , Antígenos CD4/fisiología , Linfocitos T CD4-Positivos/virología , Cuello del Útero/inmunología , Células Dendríticas/virología , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/transmisión , VIH-1 , Humanos , Técnicas In Vitro , Pruebas de Neutralización , Receptores CXCR4/antagonistas & inhibidoresRESUMEN
Chemokines play a central role in immune and inflammatory responses. It has been observed recently that certain viruses have evolved molecular piracy and mimicry mechanisms by encoding and synthesizing proteins that interfere with the normal host defense response. One such viral protein, vMIP-II, encoded by human herpesvirus 8, has been identified with in vitro antagonistic activities against CC and CXC chemokine receptors. We report here that vMIP-II has additional antagonistic activity against CX3CR1, the receptor for fractalkine. To investigate the potential therapeutic effect of this broad-spectrum chemokine antagonist, we studied the antiinflammatory activity of vMIP-II in a rat model of experimental glomerulonephritis induced by an antiglomerular basement membrane antibody. vMIP-II potently inhibited monocyte chemoattractant protein 1-, macrophage inflammatory protein 1beta-, RANTES (regulated on activation, normal T cell expressed and secreted)-, and fractalkine-induced chemotaxis of activated leukocytes isolated from nephritic glomeruli, significantly reduced leukocyte infiltration to the glomeruli, and markedly attenuated proteinuria. These results suggest that molecules encoded by some viruses may serve as useful templates for the development of antiinflammatory compounds.
Asunto(s)
Movimiento Celular/fisiología , Quimiocinas/farmacología , Glomerulonefritis/fisiopatología , Leucocitos/metabolismo , Receptores de Citocinas/antagonistas & inhibidores , Receptores del VIH/antagonistas & inhibidores , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Membrana Basal/inmunología , Unión Competitiva , Receptor 1 de Quimiocinas CX3C , Movimiento Celular/efectos de los fármacos , Quimiocinas/uso terapéutico , Quimiotaxis/efectos de los fármacos , Modelos Animales de Enfermedad , Herpesvirus Humano 8/química , Inmunohistoquímica , Inflamación/fisiopatología , Glomérulos Renales/citología , Glomérulos Renales/inmunología , Proteinuria/fisiopatología , Ratas , Ratas Endogámicas , Proteínas Virales/farmacologíaRESUMEN
The envelope glycoprotein (Env) of human immunodeficiency virus is key to viral entry of susceptible target cells and is therefore a major target for the design of vaccines and antiviral drugs. C-C chemokine receptor type 5 (CCR5)-using (R5) Env is the predominant phenotype associated with early transmission and acute infection. This study investigated the mechanism of CCR5 use and the sensitivity to CCR5 inhibitors of a panel of transmitted or early founder (T/F) Envs. The data showed that the majority of T/F Envs used CCR5 and that many also used CCR3, although less efficiently. Despite a similar ability to use wild-type CCR5, individual Envs differed significantly in their sensitivity to the CCR5 inhibitors maraviroc, CMPD-167 and SCH-412147. Inhibitor mapping experiments demonstrated that maraviroc, CMPD-167 and SCH-412147 interfered with the binding of CCR5 mAb to the C-terminal half of the second extracellular loop 2 of CCR5. Interestingly, Envs resistant to maraviroc, CMPD167 and SCH-412147 remained sensitive to TAK-779. Further studies indicated that the sensitivity of Envs to CCR5 inhibitors correlated with the molecular anatomy of CCR5 use, revealing that the inhibitor-sensitive Envs barely used the CCR5 N terminus, whereas resistant Envs showed a marked increase in its use. Taken together, these findings demonstrate that T/F R5 Envs are heterogeneous with respect to the mechanisms of CCR5 utilization. These data may have implications for therapeutic and prophylactic use of CCR5-based antiretrovirals.
Asunto(s)
Antagonistas de los Receptores CCR5 , Inhibidores de Fusión de VIH/farmacología , VIH-1/fisiología , Receptores CCR5/metabolismo , Internalización del Virus , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Amidas/farmacología , Anticuerpos Monoclonales/metabolismo , Ciclohexanos/farmacología , Humanos , Maraviroc , Unión Proteica , Pirazoles/farmacología , Compuestos de Amonio Cuaternario/farmacología , Receptores del VIH/antagonistas & inhibidores , Receptores del VIH/metabolismo , Triazoles/farmacología , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
The majority of currently available agents for the treatment of HIV-1 infection act by targeting one of several intracellular steps in the viral life cycle. Despite improvements in efficacy and tolerability, the development of viral resistance to these agents is common and significant toxicity and adherence issues still occur. For this reason the development of safe, well tolerated antiviral agents that target a novel step in the viral life cycle remains important. Viral entry into host cells affords several potential extracellular targets for antiretroviral therapy. Ibalizumab, a humanized monoclonal antibody to CD4, the primary host cellular receptor for HIV-1 entry, has been shown to block HIV-1 entry in vitro. Early clinical trials have demonstrated significant antiviral efficacy with a >1 log(10) reduction in viral load when given as monotherapy. Its long half-life, which allows weekly dosing, and its administration as an intravenous infusion differentiate it from other currently available antiretroviral agents. These properties may prove useful in allowing improved drug delivery to patients who have had difficulty adhering to daily oral regimens. Its unique mode of action reduces the risk of cross-resistance with currently available antiretroviral agents, with the potential to expand the choices available to treat drug-resistant HIV-1.
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Anticuerpos Monoclonales/uso terapéutico , Antígenos CD4/metabolismo , Infecciones por VIH/tratamiento farmacológico , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacocinética , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/farmacología , Ensayos Clínicos como Asunto , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Semivida , Humanos , Infusiones Intravenosas , Receptores del VIH/antagonistas & inhibidoresRESUMEN
Continuous specific downmodulation of CD4 receptor expression in T lymphocytes by the small molecule cyclotriazadisulfonamide (CADA) selected for the CADA-resistant human immunodeficiency virus type 1 (HIV-1) NL4.3 virus containing unique mutations in the C4 and V5 regions of gp120, likely stabilizing the CD4-binding conformation. The amino acid changes in Env were associated with decreased susceptibility to anti-CD4 monoclonal antibody treatment of the cells and with higher susceptibility of the virus to soluble CD4. In addition, the acquired ability of a CADA-resistant virus to infect cells with low CD4 expression was associated with an increased susceptibility of the virus to neutralizing antibodies from sera of several HIV-1-infected patients.
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Anticuerpos Antivirales/farmacología , Antígenos CD4/efectos de los fármacos , Farmacorresistencia Viral , VIH-1/fisiología , Sulfonamidas/farmacología , Internalización del Virus/efectos de los fármacos , Antígenos CD4/farmacología , Línea Celular , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Mutación , Receptores del VIH/antagonistas & inhibidores , Proteínas Recombinantes/farmacologíaRESUMEN
Dendritic cells (DCs) efficiently capture HIV-1 and mediate transmission to T cells, but the underlying molecular mechanism is still being debated. The C-type lectin DC-SIGN is important in HIV-1 transmission by DCs. However, various studies strongly suggest that another HIV-1 receptor on DCs is involved in the capture of HIV-1. Here we have identified syndecan-3 as a major HIV-1 attachment receptor on DCs. Syndecan-3 is a DC-specific heparan sulfate (HS) proteoglycan that captures HIV-1 through interaction with the HIV-1 envelope glycoprotein gp120. Syndecan-3 stabilizes the captured virus, enhances DC infection in cis, and promotes transmission to T cells. Removal of the HSs from the cell surface by heparinase III or by silencing syndecan-3 by siRNA partially inhibited HIV-1 transmission by immature DCs, whereas neutralizing both syndecan-3 and DC-SIGN completely abrogated HIV-1 capture and subsequent transmission. Thus, HIV-1 exploits both syndecan-3 and DC-SIGN to mediate HIV-1 transmission, and an effective microbicide should target both syndecan-3 and DC-SIGN on DCs to prevent transmission.
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Células Dendríticas/inmunología , VIH-1 , Receptores del VIH/metabolismo , Sindecano-3/metabolismo , Linfocitos T/inmunología , Internalización del Virus , Anticuerpos/farmacología , Células Dendríticas/virología , Humanos , ARN Interferente Pequeño/farmacología , Receptores del VIH/antagonistas & inhibidores , Receptores del VIH/genética , Sindecano-3/antagonistas & inhibidores , Sindecano-3/genética , Linfocitos T/virologíaRESUMEN
PURPOSE OF REVIEW: The discovery of the chemokine coreceptors CCR5 and CXCR4, essential for HIV entry, provoked over a decade of research into the mechanism of HIV entry and potential targets for treatment and prevention of HIV. This review article reports the recent published data regarding licensed and investigational HIV entry inhibitors including CCR5 and CXCR4 antagonists, fusion inhibitors and attachment inhibitors. RECENT FINDINGS: The results from the maraviroc and vicriviroc clinical trials are reported, showing the efficacy and safety of CCR5 antagonists in treatment of multidrug resistant HIV. The poor sensitivity of the original screening tropism assay is associated with virological failure in individuals mislabelled R5 tropic who receive CCR5 antagonists. Reanalyses of clinical trials using a superior screening assay are in progress. Superior immunological restoration is seen with CCR5 antagonists that may be of benefit to individuals with discordant CD4 cell response with antiretroviral therapy and to those with severe immunosuppression. SUMMARY: Research and development of HIV entry inhibitors is ongoing and provides new classes of drug that can be used in the treatment of HIV. Clinical trials will soon commence to assess the benefit of CCR5 antagonists in immune reconstitution and other therapeutic uses.
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Inhibidores de Fusión de VIH/farmacología , Inhibidores de Fusión de VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH/efectos de los fármacos , VIH/fisiología , Internalización del Virus/efectos de los fármacos , Ensayos Clínicos como Asunto , Humanos , Receptores del VIH/antagonistas & inhibidoresRESUMEN
The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (DeltaV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.
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Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Fragmentos de Péptidos/inmunología , Receptores de Quimiocina/inmunología , Receptores del VIH/antagonistas & inhibidores , Secuencia de Bases , Línea Celular , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Inhibidores de Fusión de VIH/química , Inhibidores de Fusión de VIH/inmunología , VIH-1/química , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Pruebas de Neutralización , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Receptores CCR5/química , Receptores CCR5/genética , Receptores CCR5/inmunología , Receptores de Quimiocina/química , Receptores de Quimiocina/genética , Receptores del VIH/fisiología , Replicación ViralRESUMEN
Kaposi's sarcoma-associated herpesvirus encodes a chemokine called vMIP-II. This protein displayed a broader spectrum of receptor activities than any mammalian chemokine as it bound with high affinity to a number of both CC and CXC chemokine receptors. Binding of vMIP-II, however, was not associated with the normal, rapid mobilization of calcium from intracellular stores; instead, it blocked calcium mobilization induced by endogenous chemokines. In freshly isolated human monocytes the virally encoded vMIP-II acted as a potent and efficient antagonist of chemotaxis induced by chemokines. Because vMIP-II could inhibit cell entry of human immunodeficiency virus (HIV) mediated through CCR3 and CCR5 as well as CXCR4, this protein may serve as a lead for development of broad-spectrum anti-HIV agents.