RESUMEN
Multiple studies have described a reduction in the replicative fitness of HIV-1 isolates harboring mutations that confer resistance to antiretroviral drugs. Contradictory results, however, have been obtained depending on the methodology used in each study (Quinones-Mateu, M.E., Arts, E.J., 2002. Fitness of drug resistant HIV-I: methodology and clinical implications. Drug Resist. Update 5, 224-233), affecting our understanding of the potential relationship of viral replicative fitness with HIV-1 disease. It has been demonstrated previously that both pol and env genes play a major role in HIV-1 replicative fitness of clinical isolates. Therefore, measuring clinically relevant replicative fitness using recombinant viruses where a single mutation and/or viral gene have been introduced does not seem like a reasonable approach in this era of multi-target antiretroviral therapy. A novel method was developed to measure HIV-1 replicative fitness based on recombinant viruses expressing the enhanced green fluorescent (EGFP) or the Discosoma sp. red fluorescent (DsRed2) proteins in a HIV-1NL4-3 backbone. Contrary to previous designs to analyze HIV-1 fitness, these replication competent viruses were created in an intact viral genetic background (without deleting or affecting the expression of any viral gene). This new system was used to evaluate the contribution of drug-resistance mutations in the pol and env genes to overall viral replicative fitness (in the presence and absence of drug pressure) using direct growth competition experiments. Mutations in pol showed a stronger effect on HIV-1 replicative fitness than mutations in the env gene associated with resistance to enfuvirtide, corroborating the plasticity of the later gene to accept mutations and the sensibility of the protease and reverse transcriptase enzymes to drug-associated primary mutations. In conclusion, a new protocol was used to measure HIV-1 replicative fitness in either the presence or absence of antiretroviral drugs, which may be used as a high-throughput assay to help us understand the clinical significance of viral fitness.
Asunto(s)
Productos del Gen pol/fisiología , VIH-1/fisiología , Proteínas del Envoltorio Viral/fisiología , Replicación Viral/genética , Línea Celular , Farmacorresistencia Viral/genética , Productos del Gen pol/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , VIH-1/genética , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Coloración y Etiquetado , Proteínas del Envoltorio Viral/genética , Proteína Fluorescente RojaRESUMEN
Tuberculosis (TB) is the most common life-threatening infection in human immunodeficiency virus (HIV)-infected persons and frequently occurs before the onset of severe immunodeficiency. Development of TB is associated with increased HIV type 1 (HIV-1) viral load, a fall in CD4 lymphocyte counts, and increased mortality. The aim of this study was to examine how treatment of pulmonary TB affected HIV-1 activity in HIV-1/TB-coinfected subjects with CD4 cell counts of >100 cells/mul. HIV-1/TB-coinfected subjects were recruited in Kampala, Uganda, and were monitored over time. Based upon a significant (0.5 log10 copies/ml) decrease in viral load by the end of treatment, two patient groups could be distinguished. Responders (n = 17) had more rapid resolution of anemia and pulmonary lesions on chest radiography during TB treatment. This group had a significant increase in viral load to levels not different from those at baseline 6 months after completion of TB treatment. HIV-1 viral load in nonresponders (n = 10) with TB treatment increased and at the 6 month follow-up was significantly higher than that at the time of diagnosis of TB. Compared to baseline levels, serum markers of macrophage activation including soluble CD14 decreased significantly by the end of TB treatment in responders but not in nonresponders. These data further define the impact of pulmonary TB on HIV-1 disease. HIV-1 replication during dual HIV-1/TB infection is not amenable to virologic control by treatment of TB alone. Concurrent institution of highly active antiretroviral treatment needs to be evaluated in patients dually infected with pulmonary TB and HIV-1.
Asunto(s)
Infecciones por VIH/virología , VIH-1/fisiología , Tuberculosis Pulmonar/tratamiento farmacológico , Replicación Viral/inmunología , Adulto , Biomarcadores/sangre , Comorbilidad , Femenino , Estudios de Seguimiento , Infecciones por VIH/epidemiología , Infecciones por VIH/inmunología , Humanos , Masculino , Estudios Prospectivos , ARN Viral/sangre , Tuberculosis Pulmonar/epidemiología , Tuberculosis Pulmonar/inmunología , Uganda/epidemiología , Carga Viral , Activación Viral/inmunologíaRESUMEN
OBJECTIVE: Mechanisms underlying mucosal transmission of HIV-1 are incompletely understood. We describe the anti-HIV-1 activity of human beta-defensins (hBD), small cationic molecules that provide protection at mucosal surfaces. METHODS AND RESULTS: HIV-1 induced expression of hBD-2 and -3 mRNA (but not that of hBD-1) 4- to 78-fold, respectively, above baseline in normal human oral epithelial cells. HIV-1 failed to infect these cells, even after 5 days of exposure. Recombinant hBD-1 had no antiviral activity, while rhBD-2 and rhBD-3 showed concentration-dependent inhibition of HIV-1 replication without cellular toxicity. Inhibition was greater against CXCR4-tropic than against the CCR5-tropic HIV-1 isolates. hBD-2 and hBD-3 induced an irreversible effect on virion infectivity, with electron microscopy confirming binding of hBDs to viral particles. Finally, hBD-2 and -3 induced downmodulation of the HIV-1 coreceptor CXCR4 (but not CCR5) in peripheral blood mononuclear cells and T lymphocytic cells as shown by confocal microscopy and flow cytometry. CONCLUSIONS: This study shows for the first time that HIV-1 induces beta-defensin expression in human oral epithelial cells and that beta-defensins block HIV-1 replication via a direct interaction with virions and through modulation of the CXCR4 coreceptor. These properties may be exploited as strategies for mucosal protection against HIV-1 transmission.
Asunto(s)
VIH-1/efectos de los fármacos , Mucosa Bucal/virología , Replicación Viral/efectos de los fármacos , beta-Defensinas/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Epiteliales/virología , Regulación de la Expresión Génica , VIH-1/fisiología , Humanos , Mucosa Bucal/metabolismo , ARN Mensajero/genética , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Proteínas Recombinantes/farmacología , beta-Defensinas/biosíntesis , beta-Defensinas/genéticaRESUMEN
Despite numerous studies on human immunodeficiency virus type 1 (HIV-1) fitness, many key conceptual and technical questions are still unsolved. For example, the proper system to determine virus fitness of HIV-1 is still unknown. In this study, an assay was developed to estimate HIV-1 fitness based on growth competition experiments and TaqMan real-time PCR. This novel technique was compared with several methods (i.e. virus growth kinetics, growth competition/heteroduplex-tracking analysis and single-cycle replication capacity assay) in order to analyse the impact of various genomic regions and overall genetic background on virus fitness. HIV-1 primary isolates and three different sets of recombinant viruses [i.e. recombinant clones carrying protease (PR), reverse transcriptase (RT) or the 3' end of Gag, PR and RT (3'Gag/PR/RT), sequences amplified by PCR from the same primary isolates)] were evaluated. Here, it is demonstrated that, in spite of intrinsic differences, both growth competition/TaqMan and single-cycle replication assays detected a significant reduction in HIV-1 fitness as a consequence of drug-resistant mutations in pol. However, this new assay, based on HIV-1 isolates, may be useful to quantify replicative fitness in viruses from patients treated simultaneously with antiretroviral drugs targeting different genomic regions of HIV-1 (e.g. pol and env).
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/crecimiento & desarrollo , VIH-1/fisiología , Reacción en Cadena de la Polimerasa/métodos , Inhibidores de la Transcriptasa Inversa/farmacología , Polimerasa Taq/metabolismo , Farmacorresistencia Viral/genética , Quimioterapia Combinada , Productos del Gen env/genética , Productos del Gen pol/genética , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/efectos de los fármacos , Humanos , Recombinación Genética , Replicación ViralRESUMEN
A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r = 0.93; P = 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors.