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1.
J Virol ; 87(7): 3729-40, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23325686

RESUMEN

Nef is a human immunodeficiency virus type 1 (HIV-1) auxiliary protein that plays an important role in virus replication and the onset of acquired immunodeficiency. Although known functions of Nef might explain its contribution to HIV-1-associated pathogenesis, how Nef increases virus infectivity is still an open question. In vitro, Nef-deleted viruses have a defect that prevents efficient completion of early steps of replication. We have previously shown that this restriction is not due to the absence of Nef in viral particles. Rather, a loss of function in virus-producing cells accounts for the lower infectivity of nef-deleted viruses compared to wild-type (WT) viruses. Here we used DiGE and iTRAQ to identify differences between the proteomes of WT and nef-deleted viruses. We observe that glucosidase II is enriched in WT virions, whereas Ezrin, ALG-2, CD81, and EHD4 are enriched in nef-deleted virions. Functional analysis shows that glucosidase II, ALG-2, and CD81 have no or only Nef-independent effect on infectivity. In contrast, Ezrin and EHD4 are involved in the ability of Nef to increase virus infectivity (referred to thereafter as Nef potency). Indeed, simultaneous Ezrin and EHD4 depletion in SupT1 and 293T virus-producing cells result in an ∼30 and ∼70% decrease of Nef potency, respectively. Finally, while Ezrin behaves as an inhibitory factor counteracted by Nef, EHD4 should be considered as a cofactors required by Nef to increase virus infectivity.


Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , VIH-1/genética , VIH-1/patogenicidad , Proteínas Nucleares/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/deficiencia , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Western Blotting , Electroforesis/métodos , Silenciador del Gen , Células HEK293 , VIH-1/metabolismo , Humanos , Proteómica , ARN Interferente Pequeño , Tetraspanina 28/metabolismo , Ultracentrifugación , Virión/metabolismo , alfa-Glucosidasas/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética
2.
J Virol ; 86(16): 8810-20, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22696639

RESUMEN

Despite the ability of primate lentiviruses to prevent infected cells from being reinfected, cell coinfection has occurred in the past and has shaped virus evolution by promoting the biogenesis of heterozygous virions and recombination during reverse transcription. In vitro experiments have shown that cell coinfection with HIV is more frequent than would be expected if coinfection were a random process. A possible explanation for this bias is the heterogeneity of target cells and the preferred infection of a subpopulation. To address this question, we compared the frequency of double-positive cells measured following coincubation with green fluorescent protein (GFP) and DsRed HIV reporter viruses with that of stochastic coinfection calculated as the product of the frequencies of GFP- and DsRed-positive cells upon incubation with either reporter virus. Coinfection was more frequent than would be expected on the grounds of stochastic infection, due to the underestimation of single-infection frequencies, which mathematically decreased the calculated frequency. Indeed, when cells were incubated with either reporter virus, a fraction of the cells were scored as uninfected yet harbored a silent provirus that was reactivated upon coinfection through cross talk between viral elements. When such cross talk was avoided, experimental and calculated coinfection frequencies matched, indicating random coinfection. The proportion of infected cells harboring a silent provirus was estimated from coinfection experiments and was shown to be cell type dependent but independent of the virus entry route.


Asunto(s)
VIH-1/crecimiento & desarrollo , Provirus/crecimiento & desarrollo , Línea Celular , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Coloración y Etiquetado , Proteína Fluorescente Roja
3.
Blood ; 117(13): 3559-68, 2011 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-21292773

RESUMEN

The Nef protein of HIV-1 is important for AIDS pathogenesis, but it is not targeted by current antiviral strategies. Here, we describe a single-domain antibody (sdAb) that binds to HIV-1 Nef with a high affinity (K(d) = 2 × 10(-9)M) and inhibited critical biologic activities of Nef both in vitro and in vivo. First, it interfered with the CD4 down-regulation activity of a broad panel of nef alleles through inhibition of the Nef effects on CD4 internalization from the cell surface. Second, it was able to interfere with the association of Nef with the cellular p21-activated kinase 2 as well as with the resulting inhibitory effect of Nef on actin remodeling. Third, it counteracted the Nef-dependent enhancement of virion infectivity and inhibited the positive effect of Nef on virus replication in peripheral blood mononuclear cells. Fourth, anti-Nef sdAb rescued Nef-mediated thymic CD4(+) T-cell maturation defects and peripheral CD4(+) T-cell activation in the CD4C/HIV-1(Nef) transgenic mouse model. Because all these Nef functions have been implicated in Nef effects on pathogenesis, this anti-Nef sdAb may represent an efficient tool to elucidate the molecular functions of Nef in the virus life cycle and could now help to develop new strategies for the control of AIDS.


Asunto(s)
Anticuerpos de Cadena Única/farmacología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Síndrome de Inmunodeficiencia Adquirida/terapia , Animales , Camélidos del Nuevo Mundo/inmunología , Células Cultivadas , Embrión de Mamíferos , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/metabolismo , Anticuerpos Anti-VIH/farmacología , Células HeLa , Humanos , Inmunoterapia/métodos , Células Jurkat , Ratones , Ratones Endogámicos C3H , Ratones Transgénicos , Terapia Molecular Dirigida/métodos , Unión Proteica , Estructura Terciaria de Proteína/fisiología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo
4.
Nucleic Acids Res ; 39(17): 7610-9, 2011 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-21715375

RESUMEN

Gene targeting can be achieved with lentiviral vectors delivering donor sequences along with a nuclease that creates a locus-specific double-strand break (DSB). Therapeutic applications of this system would require an appropriate control of the amount of endonuclease delivered to the target cells, and potentially toxic sustained expression must be avoided. Here, we show that the nuclease can be transferred into cells as a protein associated with a lentiviral vector particle. I-SceI, a prototypic meganuclease from yeast, was incorporated into the virions as a fusion with Vpr, an HIV accessory protein. Integration-deficient lentiviral vectors containing the donor sequences and the I-SceI fusion protein were tested in reporter cells in which targeting events were scored by the repair of a puromycin resistance gene. Molecular analysis of the targeted locus indicated a 2-fold higher frequency of the expected recombination event when the nuclease was delivered as a protein rather than encoded by a separate vector. In both systems, a proportion of clones displayed multiple integrated copies of the donor sequences, either as tandems at the targeted locus or at unrelated loci. These integration patterns were dependent upon the mode of meganuclease delivery, suggesting distinct recombination processes.


Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/genética , Marcación de Gen/métodos , Vectores Genéticos , Lentivirus/genética , Animales , Células CHO , Cricetinae , Cricetulus , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Sitios Genéticos , Células HEK293 , Humanos , Recombinación Genética , Virión/genética
5.
Cells ; 12(12)2023 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-37371112

RESUMEN

Unlocking cell secretion capacity is of paramount interest for the pharmaceutical industry focused on biologics. Here, we leveraged retention using a selective hook (RUSH) system for the identification of human osteosarcoma U2OS cell secretion modulators, through automated, high-throughput screening of small compound libraries. We created a U2OS cell line which co-expresses a variant of streptavidin addressed to the lumen-facing membrane of the endoplasmic reticulum (ER) and a recombinant anti-PD-L1 antibody. The heavy chain of the antibody was modified at its C-terminus, to which a furin cleavage site, a green fluorescent protein (GFP), and a streptavidin binding peptide (SBP) were added. We show that the U2OS cell line stably expresses the streptavidin hook and the recombinant antibody bait, which is retained in the ER through the streptavidin-SBP interaction. We further document that the addition of biotin to the culture medium triggers the antibody release from the ER, its trafficking through the Golgi where the GFP-SBP moiety is clipped off, and eventually its release in the extra cellular space, with specific antigen-binding properties. The use of this clone in screening campaigns led to the identification of lycorine as a secretion enhancer, and nigericin and tyrphostin AG-879 as secretion inhibitors. Altogether, our data support the utility of this approach for the identification of agents that could be used to improve recombinant production yields and also for a better understanding of the regulatory mechanism at work in the conventional secretion pathway.


Asunto(s)
Estreptavidina , Humanos , Proteínas Recombinantes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Línea Celular , Transporte Biológico
6.
Cells ; 11(12)2022 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-35741058

RESUMEN

As indicated by an ever-increasing number of FDA approvals, biotherapeutics constitute powerful tools for the treatment of various diseases, with monoclonal antibodies (mAbs) accounting for more than 50% of newly approved drugs between 2014 and 2018 (Walsh, 2018). The pharmaceutical industry has made great progress in developing reliable and efficient bioproduction processes to meet the demand for recombinant mAbs. Mammalian cell lines are preferred for the production of functional, complex recombinant proteins including mAbs, with Chinese hamster ovary (CHO) cells being used in most instances. Despite significant advances in cell growth control for biologics manufacturing, cellular responses to environmental changes need to be understood in order to further improve productivity. Metabolomics offers a promising approach for developing suitable strategies to unlock the full potential of cellular production. This review summarizes key findings on catabolism and anabolism for each phase of cell growth (exponential growth, the stationary phase and decline) with a focus on the principal metabolic pathways (glycolysis, the pentose phosphate pathway and the tricarboxylic acid cycle) and the families of biomolecules that impact these circuities (nucleotides, amino acids, lipids and energy-rich metabolites).


Asunto(s)
Redes y Vías Metabólicas , Metabolómica , Animales , Anticuerpos Monoclonales/metabolismo , Células CHO , Cricetinae , Cricetulus , Proteínas Recombinantes/metabolismo
7.
J Virol ; 83(2): 1093-104, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18987145

RESUMEN

The viral protein Nef contributes to the optimal infectivity of human and simian immunodeficiency viruses. The requirement for Nef during viral biogenesis particles suggests that Nef might play a role in this process. Alternatively, because Nef is incorporated into viruses, it might play a role when progeny virions reach target cells. We challenged these hypotheses by manipulating the amounts of Nef incorporated in viruses while keeping its expression level constant in producer cells. This was achieved by forcing the incorporation of Nef into viral particles by fusing a Vpr sequence to the C-terminal end of Nef. A cleavage site for the viral protease was introduced between Nef and Vpr to allow the release of Nef fragments from the fusion protein during virus maturation. We show that the resulting Nef-CS-Vpr fusion partially retains the ability of Nef to downregulate cell surface CD4 and that high amounts of Nef-CS-Vpr are incorporated into viral particles compared with what is seen for wild-type Nef. The fusion protein is processed during virion maturation and releases Nef fragments similar to those found in viruses produced in the presence of wild-type Nef. Unlike viruses produced in the presence of wild-type Nef, viruses produced in the presence of Nef-CS-Vpr do not have an increase in infectivity and are as poorly infectious as viruses produced in the absence of Nef. These findings demonstrate that the presence of Nef in viral particles is not sufficient to increase human immunodeficiency virus type 1 infectivity and suggest that Nef plays a role during the biogenesis of viral particles.


Asunto(s)
VIH-1/patogenicidad , Ensamble de Virus , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/genética , VIH-1/fisiología , Células HeLa , Humanos , Virulencia , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética
8.
J Virol ; 83(14): 7117-28, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19439470

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) Nef interferes with the endocytic machinery to modulate the cell surface expression of CD4. However, the basal trafficking of CD4 is governed by different rules in the target cells of HIV-1: whereas CD4 is rapidly internalized from the cell surface in myeloid cells, CD4 is stabilized at the plasma membrane through its interaction with the p56(lck) kinase in lymphoid cells. In this study, we showed that Nef was able to downregulate CD4 in both lymphoid and myeloid cell lines but that an increase in the internalization rate of CD4 could be observed only in lymphoid cells. Expression of p56(lck) in nonlymphoid CD4-expressing cells restores the ability of Nef in order to increase the internalization rate of CD4. Concurrent with this observation, the expression of a p56(lck)-binding-deficient mutant of CD4 in lymphoid cells abrogates the Nef-induced acceleration of CD4 internalization. We also show that the expression of Nef causes a decrease in the association of p56(lck) with cell surface-expressed CD4. Regardless of the presence of p56(lck), the downregulation of CD4 by Nef was followed by CD4 degradation. Our results imply that Nef uses distinct mechanisms to downregulate the cell surface expression levels of CD4 in either lymphoid or myeloid target cells of HIV-1.


Asunto(s)
Antígenos CD4/metabolismo , Endocitosis , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Antígenos CD4/química , Antígenos CD4/genética , Antígenos CD4/inmunología , Línea Celular , Regulación hacia Abajo , Expresión Génica , Infecciones por VIH/inmunología , Infecciones por VIH/fisiopatología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Linfocitos/inmunología , Linfocitos/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología
9.
J Virol ; 82(1): 346-57, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17959679

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) infection encounters an early block in the cells of New World monkeys because the CD4 receptor does not efficiently support HIV-1 entry. We adapted HIV-1(NL4-3) and HIV-1(KB9), two HIV-1 variants with different envelope glycoproteins, to replicate efficiently in cells expressing the CD4 and CXCR4 proteins of the common marmoset, a New World monkey. The HIV-1(NL4-3) adaptation involves three gp120 changes that result in a specific increase in affinity for the marmoset CD4 glycoprotein. The already high affinity of the HIV-1(KB9) envelope glycoproteins for marmoset CD4 did not significantly change as a result of the adaptation. Instead, changes in the gp120 variable loops and gp41 ectodomain resulted in improved replication in cells expressing the marmoset receptors. HIV-1(KB9) became relatively sensitive to neutralization by soluble CD4 and antibodies as a result of the adaptation. These results demonstrate the distinct mechanistic pathways by which the HIV-1 envelope glycoproteins can adapt to less-than-optimal CD4 molecules and provide HIV-1 variants that can overcome some of the early blocks in New World monkey cells.


Asunto(s)
Adaptación Biológica , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/crecimiento & desarrollo , VIH-1/genética , Platirrinos/virología , Animales , Antígenos CD4/metabolismo , Callithrix , Línea Celular , Proteína gp120 de Envoltorio del VIH/fisiología , Proteína gp41 de Envoltorio del VIH/fisiología , Humanos , Receptores Virales/metabolismo
10.
Front Microbiol ; 5: 232, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24904546

RESUMEN

The replication and pathogenicity of lentiviruses is crucially modulated by "auxiliary proteins" which are expressed in addition to the canonical retroviral ORFs gag, pol, and env. Strategies to inhibit the activity of such proteins are often sought and proposed as possible additions to increase efficacy of the traditional antiretroviral therapy. This requires the acquisition of an in-depth knowledge of the molecular mechanisms underlying their function. The Nef auxiliary protein is expressed uniquely by primate lentiviruses and plays an important role in virus replication in vivo and in the onset of AIDS. Among its several activities Nef enhances the intrinsic infectivity of progeny virions through a mechanism which remains today enigmatic. Here we review the current knowledge surrounding such activity and we discuss its possible role in HIV biology.

11.
Mol Aspects Med ; 31(5): 418-33, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20594957

RESUMEN

The genomes of all retroviruses encode the Gag Pol and Env structural proteins. Human and simian lentiviruses have acquired non-structural proteins among which Nef plays a major role in the evolution of viral infection towards an immunodeficiency syndrome. Indeed, in the absence of a functional nef gene, primate lentiviruses are far less pathogenic than their wild type counterparts. The multiple protein-protein interactions in which Nef is involved all contribute to explain the role played by Nef in HIV- and SIV-associated disease progression. This review summarizes common and distinct features among Nef proteins and how they contribute to increasing HIV and SIV fitness towards their respective hosts.


Asunto(s)
Productos del Gen nef/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Animales , Efecto Espectador/inmunología , VIH-1/inmunología , VIH-1/patogenicidad , VIH-2/inmunología , VIH-2/patogenicidad , Humanos , Vigilancia Inmunológica , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/patogenicidad
12.
PLoS One ; 5(10): e13249, 2010 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-20967243

RESUMEN

BACKGROUND: The entry of human immunodeficiency virus (HIV-1) into host cells involves the interaction of the viral exterior envelope glycoprotein, gp120, and receptors on the target cell. The HIV-1 receptors are CD4 and one of two chemokine receptors, CCR5 or CXCR4. METHODOLOGY/PRINCIPAL FINDINGS: We created proteoliposomes that contain CD4, the primary HIV-1 receptor, and one of the coreceptors, CXCR4. Antibodies against CD4 and CXCR4 specifically bound the proteoliposomes. CXCL12, the natural ligand for CXCR4, and the small-molecule CXCR4 antagonist, AMD3100, bound the proteoliposomes with affinities close to those associated with the binding of these molecules to cells expressing CXCR4 and CD4. The HIV-1 gp120 exterior envelope glycoprotein bound tightly to proteoliposomes expressing only CD4 and, in the presence of soluble CD4, bound weakly to proteoliposomes expressing only CXCR4. The thermal stability of CD4 and CXCR4 inserted into liposomes was examined. Thermal denaturation of CXCR4 followed second-order kinetics, with an activation energy (E(a)) of 269 kJ/mol (64.3 kcal/mol) and an inactivation temperature (T(i)) of 56°C. Thermal inactivation of CD4 exhibited a reaction order of 1.3, an E(a) of 278 kJ/mol (66.5 kcal/mol), and a T(i) of 52.2°C. The second-order denaturation kinetics of CXCR4 is unusual among G protein-coupled receptors, and may result from dimeric interactions between CXCR4 molecules. CONCLUSIONS/SIGNIFICANCE: Our studies with proteoliposomes containing the native HIV-1 receptors allowed an examination of the binding of biologically important ligands and revealed the higher-order denaturation kinetics of these receptors. CD4/CXCR4-proteoliposomes may be useful for the study of virus-target cell interactions and for the identification of inhibitors.


Asunto(s)
Antígenos CD4/metabolismo , VIH-1/metabolismo , Calor , Proteolípidos , Receptores CXCR4/metabolismo , Receptores Virales/metabolismo , Unión Proteica , Desnaturalización Proteica
13.
PLoS One ; 4(3): e5006, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19333378

RESUMEN

Secretory leukocyte protease inhibitor (SLPI) is secreted by epithelial cells in all the mucosal fluids such as saliva, cervical mucus, as well in the seminal liquid. At the physiological concentrations found in saliva, SLPI has a specific antiviral activity against HIV-1 that is related to the perturbation of the virus entry process at a stage posterior to the interaction of the viral surface glycoprotein with the CD4 receptor. Here, we confirm that recombinant SLPI is able to inhibit HIV-1 infection of primary T lymphocytes, and show that SLPI can also inhibit the transfer of HIV-1 virions from primary monocyte-derived dendritic cells to autologous T lymphocytes. At the molecular level, we show that SLPI is a ligand for the phospholipid scramblase 1 (PLSCR1) and PLSCR4, membrane proteins that are involved in the regulation of the movements of phospholipids between the inner and outer leaflets of the plasma membrane. Interestingly, we reveal that PLSCR1 and PLSCR4 also interact directly with the CD4 receptor at the cell surface of T lymphocytes. We find that the same region of the cytoplasmic domain of PLSCR1 is involved in the binding to CD4 and SLPI. Since SLPI was able to disrupt the association between PLSCR1 and CD4, our data suggest that SLPI inhibits HIV-1 infection by modulating the interaction of the CD4 receptor with PLSCRs. These interactions may constitute new targets for antiviral intervention.


Asunto(s)
Antígenos CD4/metabolismo , Proteínas de Transferencia de Fosfolípidos/fisiología , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Membrana Celular/metabolismo , Infecciones por VIH/metabolismo , Humanos , Células Jurkat , Proteínas de Transferencia de Fosfolípidos/metabolismo , Unión Proteica
14.
Apoptosis ; 12(10): 1879-92, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17653867

RESUMEN

In addition to its positive signaling function in the antigen presentation process, CD4 acts as the primary receptor for HIV-1. Contact between CD4 and the viral envelope leads to virus entry, but can also trigger apoptosis of uninfected CD4+ T-cells through a mechanism that is poorly understood. We show that Siva-1, a death domain-containing proapoptotic protein, associates with the cytoplasmic domain of CD4. This interaction is mediated by the cysteine-rich region found in the C-terminal part of the Siva-1 protein. Expression of Siva-1 specifically increases the susceptibility of both T-cell lines and unstimulated human primary CD4+ T-lymphocytes to CD4-mediated apoptosis triggered by the HIV-1 envelope, and results in activation of a caspase-dependent mitochondrial pathway. The same susceptibility is observed in T-cells expressing a truncated form of CD4 that is able to recruit Siva-1 but fails to associate with p56Lck, indicating that Siva-1 participates in a pathway independent of the p56Lck kinase activity. Altogether, these results suggest that Siva-1 might participate in the CD4-initiated signaling apoptotic pathway induced by the HIV-1 envelope in T-lymphoid cells.


Asunto(s)
Apoptosis/fisiología , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/fisiología , VIH-1 , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ligandos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Antígenos CD4/genética , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Línea Celular , VIH-1/metabolismo , VIH-1/ultraestructura , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína Sequestosoma-1 , Transducción de Señal/fisiología , Técnicas del Sistema de Dos Híbridos
15.
Traffic ; 8(1): 61-76, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17140399

RESUMEN

The HIV-1 Nef protein perturbs the trafficking of membrane proteins such as CD4 by interacting with clathrin-adaptor complexes. We previously reported that Nef alters early/recycling endosomes, but its role at the plasma membrane is poorly documented. Here, we used total internal reflection fluorescence microscopy, which restricts the analysis to a approximately 100 nm region of the adherent surface of the cells, to focus on the dynamic of Nef at the plasma membrane relative to that of clathrin. Nef colocalized both with clathrin spots (CS) that remained static at the cell surface, corresponding to clathrin-coated pits (CCPs), and with approximately 50% of CS that disappeared from the cell surface, corresponding to forming clathrin-coated vesicles (CCVs). The colocalization of Nef with clathrin required the di-leucine motif essential for Nef binding to AP complexes and was independent of CD4 expression. Furthermore, analysis of Nef mutants showed that the capacity of Nef to induce internalization and downregulation of CD4 in T lymphocytes correlated with its localization into CCPs. In conclusion, this analysis shows that Nef is recruited into CCPs and into forming CCVs at the plasma membrane, in agreement with a model in which Nef uses the clathrin-mediated endocytic pathway to induce internalization of some membrane proteins from the surface of HIV-1-infected T cells.


Asunto(s)
Clatrina/metabolismo , Productos del Gen nef/metabolismo , VIH-1/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Transporte Biológico Activo , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Membrana Celular/metabolismo , Membrana Celular/virología , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/virología , Endocitosis , Productos del Gen nef/química , Productos del Gen nef/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Microscopía Fluorescente , Proteínas Recombinantes de Fusión/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana
16.
Virology ; 353(1): 52-67, 2006 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-16808956

RESUMEN

We investigated possible interactions between HIV-1 receptor (CD4) and the main coreceptors CXCR4 and CCR5. We found that CD4 and CXCR4 coexpressed in 293T cells form a complex that can be immunoprecipitated with antibodies directed against the extracellular domain of either protein. Mutagenesis revealed that the CD4/CXCR4 interaction maps to two previously uncharacterized basic motifs in the cytoplasmic domain of CD4. HIV-1 envelope glycoprotein-mediated membrane fusion was found to be independent of the ability of CD4 and CXCR4 to interact, whether fusion was studied in a virus-cell or a cell-cell model. However, this interaction might explain the adaptation of HIV-1 to CXCR4 as an alternative to CCR5. We found that CXCR4 also interacts with the cytoplasmic domain of CD8alpha in a way that is similar to the CD4/CXCR4 interaction. The CD4/CXCR4 and CD8alpha/CXCR4 interactions may thus be involved in cellular signaling pathways shared by the CD4 and CD8alpha molecules.


Asunto(s)
Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , VIH-1/metabolismo , Fusión de Membrana/fisiología , Receptores CXCR4/fisiología , Proteínas del Envoltorio Viral/metabolismo , Antígenos CD4/química , Antígenos CD4/genética , Antígenos CD8/química , Antígenos CD8/genética , Línea Celular , Humanos , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Receptores CCR5/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo
17.
J Virol ; 76(21): 10791-800, 2002 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12368322

RESUMEN

CD4 and the chemokine receptors, CXCR4 and CCR5, serve as receptors for human immunodeficiency virus type 1 (HIV-1). Binding of the HIV-1 gp120 envelope glycoprotein to the chemokine receptors normally requires prior interaction with CD4. Mapping the determinants on gp120 for the low-affinity interaction with CXCR4 has been difficult due to the nonspecific binding of this viral glycoprotein to cell surfaces. Here we examine the binding of a panel of gp120 mutants to paramagnetic proteoliposomes displaying CXCR4 on their surfaces. We show that the gp120 beta19 strand and third variable (V3) loop contain residues important for CXCR4 interaction. Basic residues from both elements, as well as a conserved hydrophobic residue at the V3 tip, contribute to CXCR4 binding. Removal of the gp120 V1/V2 variable loops allows the envelope glycoprotein to bind CXCR4 in a CD4-independent manner. These results indicate that although some variable gp120 residues contribute to the specific binding to CCR5 or CXCR4, gp120 elements common to CXCR4- or CCR5-using strains are involved in the interaction with both coreceptors.


Asunto(s)
Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Receptores CXCR4/metabolismo , Línea Celular Transformada , Secuencia Conservada , Proteína gp120 de Envoltorio del VIH/genética , Humanos , Mutagénesis , Unión Proteica , Receptores CCR5/metabolismo
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