RESUMEN
BACKGROUND: HIV-1 capsid influences viral uncoating and nuclear import. Some capsid is detected in the nucleus but it is unclear if it has any function. We reported that the antibiotic Coumermycin-A1 (C-A1) inhibits HIV-1 integration and that a capsid mutation confers resistance to C-A1, suggesting that capsid might affect post-nuclear entry steps. RESULTS: Here we report that C-A1 inhibits HIV-1 integration in a capsid-dependent way. Using molecular docking, we identify an extended binding pocket delimited by two adjacent capsid monomers where C-A1 is predicted to bind. Isothermal titration calorimetry confirmed that C-A1 binds to hexameric capsid. Cyclosporine washout assays in Jurkat CD4+ T cells expressing engineered human TRIMCyp showed that C-A1 causes faster and greater escape from TRIMCyp restriction. Sub-cellular fractionation showed that small amounts of capsid accumulated in the nuclei of infected cells and C-A1 reduced the nuclear capsid. A105S and N74D capsid mutant viruses did not accumulate capsid in the nucleus, irrespective of C-A1 treatment. Depletion of Nup153, a nucleoporin located at the nuclear side of the nuclear pore that binds to HIV-1 capsid, made the virus less susceptible to TRIMCyp restriction, suggesting that Nup153 may help maintain some integrity of the viral core in the nucleus. Furthermore C-A1 increased binding of CPSF6, a nuclear protein, to capsid. CONCLUSIONS: Our results indicate that capsid is involved in post-nuclear entry steps preceding integration.
Asunto(s)
Proteína p24 del Núcleo del VIH/metabolismo , VIH-1/fisiología , Internalización del Virus , Aminocumarinas/metabolismo , Antivirales/metabolismo , Línea Celular , VIH-1/efectos de los fármacos , HumanosRESUMEN
HIV-2 and SIVMAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to >100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1° sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor.
Asunto(s)
Antivirales/farmacología , Cápside/microbiología , VIH-2/efectos de los fármacos , Leucocitos Mononucleares/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Integración Viral/efectos de los fármacos , Animales , Línea Celular , Células Dendríticas/efectos de los fármacos , Células Dendríticas/virología , VIH-2/genética , VIH-2/inmunología , Humanos , Leucocitos Mononucleares/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/virologíaRESUMEN
Over the last 20 years, continuous advances in the field of molecular biology have led to the development of new strategies to discover and produce monoclonal antibodies, notably by phage display.Here we describe a simple procedure for antibody selection that reduces considerably the undesired selection of non-specific antibodies, based on the use of biotinylated GST proteins fused to a target antigenic sequence. This procedure was tested on a collection of 7 different targets and resulted in the selection of a high percentage (71%) of antibodies specific for each target. This simple and effective in vitro procedure has a strong potential to replace animal immunization for the development of specific antibodies.
Asunto(s)
Anticuerpos Monoclonales/química , Glutatión Transferasa/metabolismo , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Antígenos , Biotinilación , Técnicas de Visualización de Superficie Celular , Glutatión Transferasa/química , Células HeLa , Humanos , Proteínas Recombinantes/químicaRESUMEN
Cyclophilins (Cyps), the intracellular receptors for Cyclosporine A (CsA), are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase ß1 subunit protein (Na/K-ß1) that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-ß1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA.
Asunto(s)
Ciclofilinas/metabolismo , Túbulos Renales Proximales/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Western Blotting , Células COS , Línea Celular Transformada , Chlorocebus aethiops , Ciclofilinas/genética , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Túbulos Renales Proximales/citología , Unión Proteica/efectos de los fármacos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasa Intercambiadora de Sodio-Potasio/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Cyclosporine (CsA) decreases HIV-1 infectivity by blocking HIV-1 capsid (CA) interaction with target cell cyclophilin A (CypA). Yet, HIV-1 virions produced in the presence of CsA also exhibit decreased infectivity that was previously shown to be independent of the well-characterized HIV-1 CA-CypA interaction. Here, we demonstrate that CsA decreases gp120 and gp41 incorporation into HIV-1 virions and that the fusion of these virions with susceptible target cells is impaired. This effect was not observed with HIV-1 virions pseudotyped with the vesicular stomatitis virus glycoprotein or with the amphotropic envelope protein of murine leukemia virus. It was independent of calcineurin signaling, the endoplasmic reticulum luminal protein cyclophilin B, and the long cytoplasmic tail of gp41. Thus, cyclosporine blocks HIV-1 infectivity via two independent mechanisms, the first involving HIV-1 CA in target cells and the second involving HIV-1 Env in producer cells.
Asunto(s)
Fármacos Anti-VIH/farmacología , Ciclosporina/farmacología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Virión/efectos de los fármacos , Ensamble de Virus/efectos de los fármacos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Línea Celular , Humanos , Linfocitos T/virologíaRESUMEN
Host cells impose a broad range of obstacles to the replication of retroviruses. Tetherin (also known as CD317, BST-2 or HM1.24) impedes viral release by retaining newly budded HIV-1 virions on the surface of cells. HIV-1 Vpu efficiently counteracts this restriction. Here, we show that HIV-1 Vpu induces the depletion of tetherin from cells. We demonstrate that this phenomenon correlates with the ability of Vpu to counteract the antiviral activity of both overexpressed and interferon-induced endogenous tetherin. In addition, we show that Vpu co-immunoprecipitates with tetherin and beta-TrCP in a tri-molecular complex. This interaction leads to Vpu-mediated proteasomal degradation of tetherin in a beta-TrCP2-dependent manner. Accordingly, in conditions where Vpu-beta-TrCP2-tetherin interplay was not operative, including cells stably knocked down for beta-TrCP2 expression or cells expressing a dominant negative form of beta-TrCP, the ability of Vpu to antagonize the antiviral activity of tetherin was severely impaired. Nevertheless, tetherin degradation did not account for the totality of Vpu-mediated counteraction against the antiviral factor, as binding of Vpu to tetherin was sufficient for a partial relief of the restriction. Finally, we show that the mechanism used by Vpu to induce tetherin depletion implicates the cellular ER-associated degradation (ERAD) pathway, which mediates the dislocation of ER membrane proteins into the cytosol for subsequent proteasomal degradation. In conclusion, we show that Vpu interacts with tetherin to direct its beta-TrCP2-dependent proteasomal degradation, thereby alleviating the blockade to the release of infectious virions. Identification of tetherin binding to Vpu provides a potential novel target for the development of drugs aimed at inhibiting HIV-1 replication.
Asunto(s)
Antígenos CD/metabolismo , VIH-1/patogenicidad , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Glicoproteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismo , Animales , Línea Celular , Retículo Endoplásmico , Proteínas Ligadas a GPI , Células HeLa , Humanos , Inmunoprecipitación , Interferón-alfa/metabolismo , Ratones , Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios y Motivos de Interacción de Proteínas , Virión/metabolismoAsunto(s)
Genes MHC Clase II , Antígenos HLA-D/genética , Mutagénesis Insercional , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunología , Transactivadores/deficiencia , Transactivadores/genética , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular Tumoral , Clonación Molecular , Prueba de Complementación Genética , Antígenos HLA-D/biosíntesis , Historia del Siglo XX , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/biosíntesis , Empalme del ARN , Transactivadores/biosíntesisRESUMEN
The transcription factors RFX and CIITA are major players in regulation of the expression of all classical and nonclassical major histocompatibility complex class II (MHC-II) genes. RFX nucleates the formation of a multiprotein complex, called the MHC-II enhanceosome, on MHC-II promoters. Assembly of this enhanceosome is an obligatory step for recruitment of the coactivator CIITA and thus for activation of MHC-II gene transcription. We have analyzed the function of the ankyrin repeat-containing protein RFXANK, which forms the heterotrimeric RFX complex together with RFX5 and RFXAP. We discovered that ANKRA2, the closest paralogue of RFXANK, can substitute for RFXANK in the activation of MHC-II genes and that this ability is mediated by its ankyrin repeat domain (ARD). This finding provided the basis for a high-resolution structure-function analysis of the ARD of RFXANK, which allowed us to map the RFX5 interaction domain and residues critical for assembly of the RFX complex. We also found that mutations in the fourth ankyrin repeat of RFXANK abolish assembly of the enhanceosome on MHC-II promoters in vivo but not in vitro, suggesting a new role of RFXANK in facilitating promoter occupation in the context of chromatin.
Asunto(s)
Antígenos de Histocompatibilidad Clase II , Mutagénesis , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Línea Celular , Separación Celular , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN , Citometría de Flujo , Glutatión Transferasa/metabolismo , Humanos , Inmunoprecipitación , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/química , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/química , Homología de Secuencia de Aminoácido , Transactivadores/química , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Major histocompatibility complex class II (MHCII) deficiency is a primary immunodeficiency resulting from defects in one of four different MHCII-specific transcription factors-CIITA, RFX5, RFXAP, and RFXANK. Despite this genetic heterogeneity, the phenotypical manifestations are homogeneous. It is frequently difficult to establish a definitive diagnosis of the disease on the basis of clinical and immunological criteria. Moreover, the phenotypical homogeneity precludes unambiguous identification of the regulatory gene that is affected. Identification of the four genes mutated in the disease has now allowed us to develop a rapid and straightforward diagnostic test for new MHCII-deficiency patients. This test is based on direct correction of the genetic defect by transduction of cells from patients with lentiviral vectors encoding CIITA, RFXANK, RFX5, or RFXAP. We have validated this approach by defining the molecular defects in two new patients. The RFXANK vector restored MHCII expression in a T cell line from one patient. The RFXAP vector corrected primary cells (PBL) from a second patient. Molecular analysis confirmed the presence of homozygous mutations in the RFXANK and RFXAP genes, respectively. Direct genetic correction represents a valuable tool for the diagnosis and classification of new MHCII-deficiency patients.