RESUMEN
In HIV-1 infected patients blood CD4+ T lymphocytes could be a valuable target to analyse drug resistance mutations (DRM) selected over the course of the infection. However, detection of viral resistance mutations in cellular DNA by standard genotype resistance techniques (SGRT) is suboptimal. Whole blood DNA (wbDNA) from 12 HIV-1 infected patients on ART was studied by Single Genome Sequencing (SGS) and 8 of them also by Ultradeep pyrosequencing (UDP). Results were compared with contemporary and historical DRM detected in plasma by SGRT during follow up. All the contemporary DRM detected in plasma from the viremic patients were detected by SGS and UDP (20 from 7 patients and 4 from 5 patients respectively). Out of the 67 historical DRM detected in plasma and no longer present at the time of testing, 38 (57%) were detected by SGS in 12 patients and 27 out of 46 (59%) by UDP in 8 patients. Additional DRM never reported in plasma by SGRT were detected by SGS (12 from 8 patients) and UDP (10 from 8 patients). Analysis of wbDNA from HIV-1 infected patients by SGS and UDP provides proof of concept of the value of blood DNA to investigate current and archived DRM in HIV-1 infected patients on ART.
Asunto(s)
Linfocitos T CD4-Positivos/virología , ADN Viral/genética , Farmacorresistencia Viral/genética , Infecciones por VIH/sangre , VIH-1/efectos de los fármacos , VIH-1/genética , Provirus/genética , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Análisis Mutacional de ADN , ADN Viral/sangre , Genoma Viral , Infecciones por VIH/tratamiento farmacológico , VIH-1/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Provirus/inmunología , Integración ViralRESUMEN
Long term non-progressor patients (LTNPs) are characterized by the natural control of HIV-1 infection. This control is related to host genetic, immunological and virological factors. In this work, phylogenetic analysis of the proviral nucleotide sequences in env gene from a Spanish HIV-1 LTNPs cohort identified a cluster of 6 HIV-1 controllers infected with closely-related viruses. The patients of the cluster showed common clinical and epidemiological features: drug user practices, infection in the same city (Madrid, Spain) and at the same time (late 70's-early 80's). All cluster patients displayed distinct host alleles associated with HIV control. Analysis of the virus envelope nucleotide sequences showed ancestral characteristic, lack of evolution and presence of rare amino-acids. Biological characterization of recombinant viruses with the envelope proteins from the cluster viruses showed very low replicative capacity in TZMbl and U87-CD4/CCR5 cells. The lack of clinical progression in the viral cluster patients with distinct combinations of protective host genotypes, but infected by low replicating viruses, indicate the important role of the virus in the non-progressor phenotype in these patients.
Asunto(s)
Infecciones por VIH/genética , VIH-1/genética , Replicación Viral/genética , Alelos , Progresión de la Enfermedad , Femenino , Genes env/genética , Sobrevivientes de VIH a Largo Plazo , Humanos , Masculino , Filogenia , España , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) and polytropic murine leukemia virus (MLV)-related virus are recently described human gammaretroviruses that have been associated with prostate cancer and chronic fatigue syndrome. These studies have been controversial because a number of laboratories have been unable to find evidence of XMRV in similar groups of patients or controls. Because the existence of XMRV raises many questions, we decided to study its presence in a group of patients infected with HIV-1 with a high proportion of intravenous drug use and coinfection by hepatitis C virus. METHODS: Forty HIV-1-infected patients under follow-up in our institution were screened for XMRV/MLV by nested polymerase chain reaction using primers targeting the gag and env region. Specific primers for mouse mitochondrial DNA were used to rule out contamination. RESULTS: No evidence of XMRV or polytropic MLV-related sequences was found in any sample from patients or controls. Four samples yielded polymerase chain reaction bands whose sequence corresponded to murine endogenous retroviral sequences, however, contamination with mouse cell DNA was subsequently confirmed. CONCLUSIONS: XMRV/MLV viruses do not seem to be associated with HIV-1 infection or intravenous drug use. Contamination of samples or reagents by genomic murine DNA or XMRV vectors could account for the sporadic detection of positive samples for XMRV and related agents.
Asunto(s)
Infecciones por VIH/virología , VIH-1 , Virus de la Leucemia Murina/aislamiento & purificación , Infecciones por Retroviridae/virología , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/aislamiento & purificación , Animales , ADN Viral/análisis , ADN Viral/genética , Hepatitis B/virología , Hepatitis C/virología , Humanos , Virus de la Leucemia Murina/genética , Ratones , Reacción en Cadena de la Polimerasa/métodos , Infecciones por Retroviridae/genética , España , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/virología , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/genéticaRESUMEN
Prolonged treatment of human immunodeficiency virus (HIV)-infected patients with nonnucleoside reverse transcriptase inhibitors (NNRTIs) might result in the selection of resistant mutants, the most frequent being the K103N mutation in reverse transcriptase. Resistance mutations are routinely detected by Sanger sequencing of the whole viral population, which does not detect sequence variants with frequencies below 20%. We have developed a pyrosequencing approach for the analysis of codon 103 of the HIV reverse transcriptase gene in the circulating viral population that detects variants below the limit of conventional sequencing. The method was tested with samples from 5 controls (not exposed to NNRTIs), 6 from patients exposed to NNRTIs and having a K103N mutant virus population detected by conventional sequencing, and 9 from patients previously exposed to NNRTIs that had a wild-type virus population by conventional sequencing. In 7 of 9, samples the mutation could not be detected by either the standard assay or pyrosequencing, while in 2 samples persistence of the mutation could be detected by pyrosequencing. The method might be of practical use in detecting minority variants of HIV in the clinical setting, in epidemiological studies with large numbers of samples, or as a complement to more complex approaches.
Asunto(s)
Farmacorresistencia Viral , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/genética , VIH/genética , Mutación Missense , Análisis de Secuencia de ADN/métodos , Adolescente , Adulto , Niño , Femenino , VIH/aislamiento & purificación , Humanos , Masculino , Pruebas de Sensibilidad Microbiana/métodos , Persona de Mediana Edad , Adulto JovenRESUMEN
The development of compounds with strong affinity for the receptor DC-SIGN is a topic of remarkable interest due to the role that this lectin plays in several pathogen infection processes and in the modulation of the immune response. DC-SIGN recognizes mannosylated and fucosylated oligosaccharides in a multivalent manner. Therefore, multivalent carbohydrate systems are required to interact in an efficient manner with this receptor and compete with the natural ligands. We have previously demonstrated that linear pseudodi- and pseudotrisaccharides are adequate ligands for DC-SIGN. In this work, we show that multivalent presentations of these glycomimetics based on polyester dendrons and dendrimers lead to very potent inhibitors (in the nanomolar range) of cell infection by Ebola pseudotyped viral particles by blocking DC-SIGN receptor. Furthermore, SPR model experiments confirm that the described multivalent glycomimetic compounds compete in a very efficient manner with polymannosylated ligands for binding to DC-SIGN.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Carbohidratos/química , Carbohidratos/farmacología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Dendrímeros/química , Dendrímeros/farmacología , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Lectinas Tipo C/antagonistas & inhibidores , Receptores de Superficie Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Ebolavirus/efectos de los fármacos , Expresión Génica , Humanos , Células Jurkat , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Unión Proteica , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
We present an HIV-1-infected patient with a profile of transmitted drug resistance (RT M41L, E44D, V118I, L210W, T215D) sustained during more than 10 years in the absence of treatment. Clonal analysis of different plasma and cellular samples within this period did not reveal any reversion to the wild-type genotype.
Asunto(s)
Sustitución de Aminoácidos/genética , Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/genética , VIH-1/genética , Mutación Missense , Adulto , Análisis por Conglomerados , Femenino , VIH-1/clasificación , VIH-1/efectos de los fármacos , VIH-1/aislamiento & purificación , Humanos , Filogenia , ARN Viral/genéticaRESUMEN
New commercial techniques for determination of the viral load (VL) in plasma are able to detect as few as 20 copies of HIV-1 RNA/ml. The relevance of this new technical threshold is uncertain. Upon multivariate analysis, factors associated with detection of VLs between 20 and 49 copies/ml by the Cobas TaqMan HIV-1 v2.0 assay in an HIV clinic were the basal VL and time on antiretroviral therapy.
Asunto(s)
Terapia Antirretroviral Altamente Activa , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Carga Viral , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Plasma/virología , ARN Viral/sangre , Juego de Reactivos para Diagnóstico , Factores de TiempoRESUMEN
Melanoma is the most aggressive skin cancer once metastasis begins; therefore, it is important to characterize the molecular players involved in melanoma dissemination. The chemokine receptor CXCR4 and the membrane-bound metalloproteinase MT1-MMP are expressed on melanoma cells and represent candidate molecules for the control of metastasis. Using human melanoma transfectants that either overexpress or silence CXCR4 or MT1-MMP, or that have a combination of overexpression and interference of these proteins, we show that CXCR4 and MT1-MMP coordinate their activities at different steps along melanoma cell metastasis into the lungs. Results from in vivo xenograft mouse models of melanoma lung colonization and mice survival and short-term, homing nested polymerase chain reaction experiments from lung samples indicated that CXCR4 is required at early phases of melanoma cell arrival in the lungs. In contrast, MT1-MMP is not needed for these initial steps but promotes subsequent invasion and dissemination of the tumor with CXCR4. Investigation of potential cross talk between CXCR4 and MT1-MMP revealed that MT1-MMP accumulates intracellularly after melanoma cell stimulation with the CXCR4 ligand CXCL12, and that this process involves the activation of the Rac-Erk1/2 pathway. Subsequent to cell contact with specific basement membrane proteins, MT1-MMP redistributes to the cell membrane in a phosphatidylinositol 3-kinase-dependent manner. These results suggest that combination therapies that target CXCR4 and MT1-MMP should improve the limitations of the current therapies for metastatic melanoma.
Asunto(s)
Neoplasias Pulmonares/secundario , Metaloproteinasa 14 de la Matriz/metabolismo , Melanoma/secundario , Invasividad Neoplásica , Receptores CXCR4/metabolismo , Neoplasias Cutáneas/patología , Animales , Western Blotting , Adhesión Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular , Citometría de Flujo , Humanos , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Ratones , Ratones SCID , Fosfatidilinositol 3-Quinasas/metabolismo , Reacción en Cadena de la Polimerasa , Receptor Cross-Talk/fisiología , Transducción de Señal/fisiología , Neoplasias Cutáneas/metabolismo , TransfecciónRESUMEN
Human immunodeficiency virus (HIV)-1 infection requires envelope (Env) glycoprotein gp120-induced clustering of CD4 and coreceptors (CCR5 or CXCR4) on the cell surface; this enables Env gp41 activation and formation of a complex that mediates fusion between Env-containing and target-cell membranes. Kinetic studies show that viral receptors are actively transported to the Env-receptor interface in a process that depends on plasma membrane composition and the actin cytoskeleton. The mechanisms by which HIV-1 induces F-actin rearrangement in the target cell remain largely unknown. Here, we show that CD4 and the coreceptors interact with the actin-binding protein filamin-A, whose binding to HIV-1 receptors regulates their clustering on the cell surface. We found that gp120 binding to cell receptors induces transient cofilin-phosphorylation inactivation through a RhoA-ROCK-dependent mechanism. Blockade of filamin-A interaction with CD4 and/or coreceptors inhibits gp120-induced RhoA activation and cofilin inactivation. Our results thus identify filamin-A as an adaptor protein that links HIV-1 receptors to the actin cytoskeleton remodelling machinery, which may facilitate virus infection.
Asunto(s)
Actinas/fisiología , Antígenos CD4/metabolismo , Proteínas Contráctiles/fisiología , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Proteínas de Microfilamentos/fisiología , Factores Despolimerizantes de la Actina/metabolismo , Secuencia de Aminoácidos , Línea Celular , Filaminas , VIH-1/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The design and preparation of carbohydrate ligands for DC-SIGN is a topic of high interest because of the role played by this C-type lectin in immunity and infection processes. The low chemical stability of carbohydrates against enzymatic hydrolysis by glycosylases has stimulated the search for new alternatives more stable in vivo. Herein, we present a good alternative for a DC-SIGN ligand based on a mannobioside mimic with a higher enzymatic stability than the corresponding disaccharide. NMR and docking studies have been performed to study the interaction of this mimic with DC-SIGN in solution demonstrating that this pseudomannobioside is a good ligand for this lectin. In vitro studies using an infection model with Ebola pseudotyped virus demonstrates that this compound presents an antiviral activity even better than the corresponding disaccharide and could be an interesting ligand to prepare multivalent systems with higher affinities for DC-SIGN with potential biomedical applications.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Moléculas de Adhesión Celular/química , Lectinas Tipo C/química , Mananos/química , Mananos/farmacología , Imitación Molecular , Receptores de Superficie Celular/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Resonancia Magnética Nuclear Biomolecular , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin [CLEC4G]) is a C-type lectin encoded within the liver/lymph node-specific intercellular adhesion molecule-3-grabbing nonintegrin (L-SIGN)/dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)/CD23 gene cluster. LSECtin expression has been previously described as restricted to sinusoidal endothelial cells of the liver and lymph node. We now report LSECtin expression in human peripheral blood and thymic dendritic cells isolated ex vivo. LSECtin is also detected in monocyte-derived macrophages and dendritic cells at the RNA and protein level. In vitro, interleukin-4 (IL-4) induces the expression of 3 LSECtin alternatively spliced isoforms, including a potentially soluble form (Delta 2 isoform) and a shorter version of the prototypic molecule (Delta3/4 isoform). LSECtin functions as a pathogen receptor, because its expression confers Ebola virus-binding capacity to leukemic cells. Sugar-binding studies indicate that LSECtin specifically recognizes N-acetyl-glucosamine, whereas no LSECtin binding to Mannan- or N-acetyl-galactosamine-containing matrices are observed. Antibody or ligand-mediated engagement triggers a rapid internalization of LSECtin,which is dependent on tyrosine and diglutamic-containing motifs within the cytoplasmic tail. Therefore, LSECtin is a pathogen-associated molecular pattern receptor in human myeloid cells. In addition, our results suggest that LSECtin participates in antigen uptake and internalization, and might be a suitable target molecule in vaccination strategies.