RESUMEN
The clinical management of HIV-1 infection has benefited enormously from molecular characterization of drug resistance as well as determination of the viral phenotype in vitro. HIV-1 infected individuals on HAART are currently monitored for the development of drug resistance variants allowing clinicians to redesign drug regimens. An understanding of the molecular basis of the evolution of drug resistance in vivo allows the improvement of the drugs as well as in vitro evaluation of new antiviral compounds alone or in combination with those currently approved. New findings suggest that viral envelopes could be a target to inhibit infection and replication. Therefore the generation of a recombinant virus assay (RVA) to allow the phenotypic determination of drug resistance against entry inhibitors (EI) is anticipated. We constructed an env-deleted clone of HIV-1 using the molecular clone NL-4.3. PCR amplified complete envelope genes (NL-4.3, BaL, primary envelope-genes) were ligated in vitro with a deletion clone (pNL-deltaK) and PM1-cells, supporting the replication of R5- and X4-tropic viruses, were transfected. Determination of co-receptor usage of the harvested recombinant virus-swarm revealed no difference compared to the molecular clones derived individually from three different patients. These results clearly show that an envelope-based RVA is practicable to monitor HIV-co-receptor usage at a given time point. Furthermore, this assay will allow to monitor resistance development against existing and future entry inhibitors and will aid to improve the management of HIV-therapy.
Asunto(s)
Bioensayo , Farmacorresistencia Viral/genética , VIH-1/fisiología , Técnicas Microbiológicas , Receptores del VIH/metabolismo , Proteínas del Envoltorio Viral/genética , Terapia Antirretroviral Altamente Activa , Línea Celular , Electroporación , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Cinética , Linfocitos/virología , Fenotipo , Recombinación Genética , Proteínas del Envoltorio Viral/metabolismo , Replicación ViralRESUMEN
Increased rates of T-cell apoptosis have been detected in human immunodeficiency virus (HIV)-infected individuals and in the simian immunodeficiency model (SIV) for AIDS research. We have infected macaques with virulent SIV or SIV/HIV hybrid viruses (SHIV) of different pathogenic potentials to study the early kinetics of apoptosis in this model. Animals infected with SIV showed an increased degree of apoptosis in their peripheral blood mononuclear cells as early as 8 weeks after virus inoculation. Apoptotic cells were detected in the CD4 and CD8 cell populations of infected animals. In contrast, apathogenic SHIV did not lead to increased lymphocyte apoptosis and moderately pathogenic SHIV induced only transient apoptosis. T-cell death was temporally linked to viral replication in vivo. Furthermore, lymphocyte apoptosis in infected macaques was associated with impaired proliferative responses of helper T-cells and with CD4 cell depletion. The monkey model described here provides the opportunity for testing early therapeutic interventions to prevent virus-induced programmed cell death and the subsequent onset of AIDS.
