RESUMEN
The gp120 envelope glycoprotein of primary human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and the CCR5 chemokine receptor on the target cell. Previously, we adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for CD4-independent replication were limited to the V2 loop-V1/V2 stem. Here we show that elimination of a single glycosylation site at asparagine 197 in the V1/V2 stem is sufficient for CD4-independent gp120 binding to CCR5 and for HIV-1 entry into CD4-negative cells expressing CCR5. Deletion of the V1/V2 loops also allowed CD4-independent viral entry and gp120 binding to CCR5. The binding of the wild-type ADA gp120 to CCR5 was less dependent upon CD4 at 4 degrees C than at 37 degrees C. In the absence of the V1/V2 loops, neither removal of the N-linked carbohydrate at asparagine 197 nor lowering of the temperature increased the CD4-independent phenotypes. A CCR5-binding conformation of gp120, achieved by CD4 interaction or by modification of temperature, glycosylation, or variable loops, was preferentially recognized by the monoclonal antibody 48d. These results suggest that the CCR5-binding region of gp120 is occluded by the V1/V2 variable loops, the position of which can be modulated by temperature, CD4 binding, or an N-linked glycan in the V1/V2 stem.
Asunto(s)
Antígenos CD4/fisiología , Proteína gp120 de Envoltorio del VIH/química , VIH-1/fisiología , Anticuerpos Monoclonales/inmunología , Línea Celular , Glicosilación , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/fisiología , Humanos , Mutagénesis Sitio-Dirigida , Receptores CCR5/metabolismo , Relación Estructura-Actividad , TemperaturaRESUMEN
Naturally occurring human immunodeficiency virus (HIV-1) variants require the presence of CD4 and specific chemokine receptors to enter a cell. In the laboratory, HIV-1 variants that are capable of bypassing CD4 and utilizing only the CCR5 chemokine receptor for virus entry have been generated. Here we report that these CD4-independent viruses are significantly more sensitive to neutralization by soluble CD4 and a variety of antibodies. The same amino acid changes in the HIV-1 gp120 envelope glycoprotein determined CD4 independence and neutralization sensitivity. The CD4-independent envelope glycoproteins exhibited higher affinity for antibodies against CD4-induced gp120 epitopes but not other neutralizing ligands. The CD4-independent envelope glycoproteins did not exhibit increased lability relative to the wild-type envelope glycoproteins. The utilization of two receptors apparently allows HIV-1 to maintain a more neutralization-resistant state prior to engaging CD4 on the target cell, explaining the rarity of CD4 independence in wild-type HIV-1.
Asunto(s)
Antígenos CD4/metabolismo , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , VIH-1/metabolismo , Secuencia de Aminoácidos , Antígenos CD4/farmacología , Línea Celular , Células Gigantes/fisiología , Anticuerpos Anti-VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/genética , Humanos , Ligandos , Datos de Secuencia Molecular , Pruebas de Neutralización , Receptores CCR5/metabolismo , Proteínas Recombinantes/farmacología , Transfección , Esparcimiento de VirusRESUMEN
The functional unit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins is a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The lability of intersubunit interactions has hindered the production and characterization of soluble, homogeneous envelope glycoprotein trimers. Here we report three modifications that stabilize soluble forms of HIV-1 envelope glycoprotein trimers: disruption of the proteolytic cleavage site between gp120 and gp41, introduction of cysteines that form intersubunit disulfide bonds, and addition of GCN4 trimeric helices. Characterization of these secreted glycoproteins by immunologic and biophysical methods indicates that these stable trimers retain structural integrity. The efficacy of the GCN4 sequences in stabilizing the trimers, the formation of intersubunit disulfide bonds between appropriately placed cysteines, and the ability of the trimers to interact with a helical, C-terminal gp41 peptide (DP178) support a model in which the N-terminal gp41 coiled coil exists in the envelope glycoprotein precursor and contributes to intersubunit interactions within the trimer. The availability of stable, soluble HIV-1 envelope glycoprotein trimers should expedite progress in understanding the structure and function of the virion envelope glycoprotein spikes.
Asunto(s)
Productos del Gen env/química , Productos del Gen env/genética , VIH-1/química , Secuencia de Aminoácidos , Biopolímeros , Centrifugación por Gradiente de Densidad , Cromatografía/métodos , Enfuvirtida , Productos del Gen env/inmunología , Productos del Gen env/metabolismo , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Pruebas de Precipitina , Receptores CCR5/metabolismo , SolubilidadRESUMEN
Seven-transmembrane segment, G protein-coupled receptors (GPCRs) play important roles in many biological processes in which pharmaceutical intervention may be useful. High level expression and native purification of GPCRs are important steps in the biochemical and structural characterization of these molecules. Here, we describe enhanced mammalian cell expression and purification of a codon-optimized variant of the chemokine receptor CCR5, a GPCR that plays a central role in the entry of the human immunodeficiency virus-1 (HIV-1) into immune cells. CCR5 could be solubilized in its native state as determined by its ability to be precipitated by 2D7, a conformation-dependent anti-CCR5 antibody, and by the HIV-1 gp120 envelope glycoprotein. The 2D7 antibody recognized immature and mature forms of CCR5 equally, whereas gp120 preferentially recognized the mature form, a result that underscores a role for posttranslational modification of CCR5 in its HIV-1 coreceptor function. The methods described herein contribute to the analysis of CCR5 and are likely to be applicable to many other GPCRs.
Asunto(s)
Receptores CCR5/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Unión Proteica , Receptores CCR5/aislamiento & purificación , Receptores CCR5/metabolismoRESUMEN
The gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and chemokine receptors on the target cell. Primary, clinical HIV-1 isolates require interaction with CD4 to allow gp120 to bind the CCR5 chemokine receptor efficiently. We adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for the adaptation were limited to alteration of glycosylation addition sites in the V2 loop-V1-V2 stem. The gp120 glycoproteins of the adapted viruses bound CCR5 directly, without prior interaction with CD4. Thus, a major function of CD4 binding in the entry of primary HIV-1 isolates can be bypassed by changes in the gp120 V1-V2 elements, which allow the envelope glycoproteins to assume a conformation competent for CCR5 binding.
Asunto(s)
Antígenos CD4/fisiología , VIH-1/fisiología , Receptores CCR5/fisiología , Replicación Viral , Animales , Perros , Proteína gp120 de Envoltorio del VIH/fisiología , Células HeLa , HumanosRESUMEN
Chemokine receptors and related seven-transmembrane-segment (7TMS) receptors serve as coreceptors for entry of human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV) into target cells. Each of these otherwise diverse coreceptors contains an N-terminal region that is acidic and tyrosine rich. Here, we show that the chemokine receptor CCR5, a principal HIV-1 coreceptor, is posttranslationally modified by O-linked glycosylation and by sulfation of its N-terminal tyrosines. Sulfated tyrosines contribute to the binding of CCR5 to MIP-1 alpha, MIP-1 beta, and HIV-1 gp120/CD4 complexes and to the ability of HIV-1 to enter cells expressing CCR5 and CD4. CXCR4, another important HIV-1 coreceptor, is also sulfated. Tyrosine sulfation may contribute to the natural function of many 7TMS receptors and may be a modification common to primate immunodeficiency virus coreceptors.
Asunto(s)
VIH-1/fisiología , Procesamiento Proteico-Postraduccional , Receptores CCR5/química , Tirosina/análogos & derivados , Secuencia de Aminoácidos , Animales , Antígenos CD4/metabolismo , Conformación de Carbohidratos , Células Cultivadas , Quimiocina CCL4 , Cloratos/farmacología , Perros , Glicosilación , Proteína gp120 de Envoltorio del VIH/metabolismo , Células HeLa , Humanos , Proteínas Inflamatorias de Macrófagos/metabolismo , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores CCR5/fisiología , Receptores CXCR4/química , Receptores CXCR4/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sulfotransferasas/metabolismo , Células Tumorales Cultivadas , Tirosina/biosíntesis , Tirosina/fisiologíaRESUMEN
Biochemical and structural studies of fragments of the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein have demonstrated that the molecular contacts between alpha helices allow the formation of a trimeric coiled coil. By introducing cysteine residues into specific locations along these alpha helices, the normally labile HIV-1 gp160 envelope glycoprotein was converted into a stable disulfide-linked oligomer. Although proteolytic cleavage into gp120 and gp41 glycoproteins was largely blocked, the disulfide-linked oligomer was efficiently transported to the cell surface and was recognized by a series of conformationally dependent antibodies. The pattern of hetero-oligomer formation between this construct and an analogous construct lacking portions of the gp120 variable loops and of the gp41 cytoplasmic tail demonstrates that these oligomers are trimers. These results support the relevance of the proposed gp41 structure and intersubunit contacts to the native, complete HIV-1 envelope glycoprotein. Disulfide-mediated stabilization of the labile HIV-1 envelope glycoprotein oligomer, which has been suggested to possess advantages as an immunogen, may assist attempts to develop vaccines.
Asunto(s)
Cisteína/química , Disulfuros , Proteína gp41 de Envoltorio del VIH/química , VIH-1/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular Transformada , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación ProteicaRESUMEN
The "disease-specific" (dsp) region next to the hrp gene cluster of Erwinia amylovora is required for pathogenicity but not for elicitation of the hypersensitive reaction. A 6.6-kb apparent operon, dspEF, was found responsible for this phenotype. The operon contains genes dspE and dspF and is positively regulated by hrpL. A BLAST search revealed similarity in the dspE gene to a partial sequence of the avrE locus of Pseudomonas syringae pathovar tomato. The entire avrE locus was sequenced. Homologs of dspE and dspF were found in juxtaposed operons and were designated avrE and avrF. Introduced on a plasmid, the dspEF locus rendered P. syringae pv. glycinea race 4 avirulent on soybean. An E. amylovora dspE mutant, however, elicited a hypersensitive reaction in soybean. The avrE locus in trans restored pathogenicity to dspE strains of E. amylovora, although restored strains were low in virulence. DspE and AvrE are large (198 kDa and 195 kDa) and hydrophilic. DspF and AvrF are small (16 kDa and 14 kDa) and acidic with predicted amphipathic alpha helices in their C termini; they resemble chaperones for virulence factors secreted by type III secretion systems of animal pathogens.
Asunto(s)
Erwinia/genética , Enfermedades de las Plantas/genética , Pseudomonas/genética , Elementos Transponibles de ADN , Erwinia/patogenicidad , Solanum lycopersicum , Datos de Secuencia Molecular , Mutagénesis , Operón , Plantas Tóxicas , Pseudomonas/patogenicidad , Glycine max , NicotianaRESUMEN
The human CXCR-4 molecule serves as a second receptor for primary, T-cell-tropic, and laboratory-adapted human immunodeficiency virus type 1 (HIV-1) isolates. Here we show that murine CXCR-4 can support the entry of some of these HIV-1 isolates. Differences between mouse and human CXCR-4 in the ability to function as an HIV-1 receptor are determined by sequences in the second extracellular loop of the CXCR-4 protein.