RESUMO
Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydroosmotic equilibrium and, to a lesser extent, on vasomotricity. Drugs currently acting on this receptor are analogs of the natural neuropeptide, arginine vasopressin (AVP), and hence are competitive ligands. Peptides that reproduce specific sequences of a given receptor have lately been reported to interfere with its action, and if such molecules arise from regions remote from the binding site they would be anticipated to exhibit noncompetitive antagonism, but this has yet to be shown for V2R. Six peptides reproducing juxtamembranous regions of V2R were designed and screened; the most effective peptide, cravky (labeled VRQ397), was characterized. VRQ397 was potent (IC(50) = 0.69 +/- 0.25 nM) and fully effective in inhibiting V2R-dependent physiological function, specifically desmopressin-L-desamino-8-arginine-vasopressin (DDAVP)-induced cremasteric vasorelaxation; this physiological functional assay was utilized to avoid overlooking interference of specific signaling events. A dose-response profile revealed a noncompetitive property of VRQ397; correspondingly, VRQ397 bound specifically to V2R-expressing cells could not displace its natural ligand, AVP, but modulated AVP binding kinetics (dissociation rate). Specificity of VRQ397 was further confirmed by its inability to bind to homologous V1 and oxytocin receptors and its inefficacy to alter responses to stimulation of these receptors. VRQ397 exhibited pharmacological permissiveness on V2R-induced signals, as it inhibited DDAVP-induced PGI(2) generation but not that of cAMP or recruitment of beta-arrestin2. Consistent with in vitro and ex vivo effects as a V2R antagonist, VRQ397 displayed anticipated in vivo aquaretic efficacy. We hereby describe the discovery of a first potent noncompetitive antagonist of V2R, which exhibits functional selectivity, in line with properties of a negative allosteric modulator.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos , Antagonistas de Hormônios/farmacologia , Músculo Liso/efeitos dos fármacos , Miométrio/efeitos dos fármacos , Oligopeptídeos/farmacologia , Bexiga Urinária/efeitos dos fármacos , 6-Cetoprostaglandina F1 alfa/metabolismo , Regulação Alostérica , Animais , Arginina Vasopressina/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Desamino Arginina Vasopressina/metabolismo , Diurese/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Antagonistas de Hormônios/metabolismo , Humanos , Técnicas In Vitro , Ligantes , Masculino , Camundongos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/metabolismo , Miométrio/metabolismo , Oligopeptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Bexiga Urinária/metabolismoRESUMO
The human cytomegalovirus encodes a beta-chemokine receptor (US28) that is distantly related to the human chemokine receptors CCR5 and CXCR4, which also serve as cofactors for the entry into cells of human immunodeficiency virus-type 1 (HIV-1). Like CCR5, US28 allowed infection of CD4-positive human cell lines by primary isolates of HIV-1 and HIV-2, as well as fusion of these cell lines with cells expressing the viral envelope proteins. In addition, US28 mediated infection by cell line-adapted HIV-1 for which CXCR4 was an entry cofactor.
Assuntos
Citomegalovirus/genética , HIV-1/fisiologia , HIV-2/fisiologia , Receptores de Quimiocinas , Receptores de Citocinas/fisiologia , Receptores de HIV/fisiologia , Proteínas Virais/fisiologia , Infecções Oportunistas Relacionadas com a AIDS/virologia , Sequência de Aminoácidos , Fusão Celular , Quimiocinas , Técnicas de Cocultura , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/virologia , Células Gigantes , Infecções por HIV/virologia , Células HeLa , Humanos , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Receptores CCR2 , Receptores CCR5 , Receptores CXCR4 , Receptores de Citocinas/genética , Receptores de HIV/genética , Transfecção , Células Tumorais Cultivadas , Proteínas Virais/genéticaRESUMO
BACKGROUND: The chemokine receptor CXCR4 (a receptor for the Cys-X-Cys class of chemokines) is a CD4-associated coreceptor for T-cell-tropic strains of human immunodeficiency virus 1 (HIV-1) and represents a target for antiviral therapy. Infection by T-tropic HIV-1 can be blocked by stromal-cell-derived factor-1 (SDF-1), the natural ligand of CXCR4. The broad variety of cells expressing CXCR4 and the perturbations observed in mice deficient for SDF-1 suggest that antiviral compounds antagonizing the signalling activity of CXCR4 might have severe side effects in vivo. Compounds that interfere selectively with HIV entry and not with SDF-1 signalling would therefore be useful. RESULTS: A series of peptides, each of 13 residues, spanning the whole SDF-1alpha sequence were tested for their ability to block HIV-1 infection. The antiviral and signalling properties of SDF-1 were retained by a peptide corresponding to its amino terminus. Removal of the first two residues resulted in an antiviral antagonist of the SDF-1-CXCR4 signalling pathway. We prepared 234 single-substitution analogues and identified one antiviral analogue that had drastically reduced agonistic or antagonistic properties. The antiviral peptides competed with the monoclonal antibody 12G5 for CXCR4 binding. Their antiviral activity seems to be due to receptor occupancy rather than induction of receptor endocytosis. CONCLUSIONS: The amino terminus of the SDF-1 chemokine is sufficient for signal transduction via CXCR4 and for inhibition of HIV-1 entry, but these activities could be dissociated in a peptide analogue. This peptide represents a lead molecule for the design of low molecular weight antiviral drugs.
Assuntos
Fármacos Anti-HIV/farmacologia , Quimiocinas CXC/farmacologia , HIV-1/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Fármacos Anti-HIV/química , Quimiocina CXCL12 , Quimiocinas CXC/química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Infecções por HIV/prevenção & controle , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Receptores CXCR4/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Chemokine receptors recruit the multifunctional scaffolding protein beta arrestin in response to binding of their chemokine ligands. Given that arrestin recruitment represents a signaling axis that is in part independent from G-protein signaling, it has become a hallmark of G protein-coupled receptor functional selectivity. Therefore, quantification of arrestin recruitment has become a requirement for the delineation of chemokine and drug candidate activity along different signaling axes. Bioluminescence resonance energy transfer (BRET) techniques provide methodology for such quantification that can reveal differences between nonredundant chemokines binding the same receptor, and that can be upscaled for high-throughput testing. We here provide protocols for the careful setup of BRET-based arrestin recruitment assays, and examples for the application of such systems in dose-response or time-course experiments. Suggestions are given for troubleshooting, optimizing test systems, and the interpretation of results obtained with BRET-based assays, which indeed yield an intricate blend of quantitative and qualitative information.
Assuntos
Arrestinas/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Receptores de Quimiocinas/metabolismo , Arrestinas/análise , Quimiocinas/metabolismo , Quimiocinas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Ligantes , Mutação , Mapeamento de Interação de Proteínas/métodos , Receptores de Quimiocinas/análise , Receptores de Quimiocinas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Razão Sinal-RuídoRESUMO
In addition to its hemostatic functions, factor (F)VIIa exhibits cell proliferative properties as seen in angiogenesis and tumor growth. A role for tissue factor (TF) and protease-activated receptors (PAR)-1 and -2 in cell proliferation remain to be clarified. We tested the hypothesis that FVIIa induces cell proliferation by a mechanism involving TF and PAR-2. Human recombinant FVIIa induced cell proliferation of human BOSC23 cells transfected with plasmid containing human TF DNA sequence. Because DNA primase 1 (PRIM1) plays an essential role in cell proliferation, we used the cloned PRIM1 promoter upstream of the reporter gene chloramphenicol acetyl transferase (CAT) to elucidate the mode of action of FVIIa. FVIIa evoked a dose-dependent increase in cell proliferation and PRIM1 induction, which were markedly potentiated (4-5-fold) by the presence of TF and abrogated by TF antisense oligonucleotide. PRIM1 induction by FVIIa was also abolished by PAR-2 but not by PAR-1 antisense. In contrast, thrombin induced a small increase in CAT activity which was unaffected by TF, but was prevented only by PAR-1 antisense as well as the thrombin inhibitor hirudin. Proliferative properties of FVIIa were associated with a TF-dependent increase in intracellular calcium and were mediated by a concordant phosphorylation of p44/42 MAP kinase. In conclusion, data reveal that FVIIa induces PRIM1 and ensuing cellular proliferation via a TF- and of the PARs entirely PAR-2-dependent pathway, in distinction to that of thrombin which is PAR-1-dependent and TF-independent. We speculate that FVIIa-TF-PAR-2 inhibitors may be effective in suppressing cell proliferation.
Assuntos
Fator VIIa/metabolismo , Receptor PAR-2/metabolismo , Tromboplastina/fisiologia , Northern Blotting , Western Blotting , Linhagem Celular , Proliferação de Células , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , DNA/química , DNA Primase/metabolismo , Relação Dose-Resposta a Droga , Genes Reporter , Hirudinas/metabolismo , Humanos , Oligonucleotídeos Antissenso/química , Fosforilação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Receptor PAR-1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Trombina/metabolismo , Tromboplastina/metabolismo , Timidina/química , Fatores de Tempo , TransfecçãoRESUMO
The discovery that chemokine receptors act as cofactors indispensable for HIV entry into target cells identified new targets for anti-retroviral therapy. However, much remains to be learned about the nature of their physiological role in the organism, as well as the molecular details of viral entry. The multitude of different receptors permitting HIV entry in vitro and their respective roles in vivo for entry, as well as their implication in distinct pathogenic events have added further complexity to this field of research. This review summarizes knowledge on HIV-coreceptors, their role under normal physiological conditions as well as in HIV pathogenesis and its implications on the development of concepts for the use of coreceptor targeting therapeutic approaches. An overview over antiviral ligands of chemokine receptors reported so far, as well as alternative strategies of antiviral interventions involving chemokine receptors is given.
Assuntos
Fármacos Anti-HIV/farmacologia , Antivirais/farmacologia , Infecções por HIV/tratamento farmacológico , Receptores de Quimiocinas/efeitos dos fármacos , Retroviridae/efeitos dos fármacos , Animais , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HumanosRESUMO
Epidermal Langerhans cells (ELC) are definitively primed to differentiate into dendritic cells (DC). It is unknown at what stage of monocyte development this priming occurs. In a culture system characterized by low paracrine stimulation, i.e. Iscove's modified Dulbecco medium (IMDM) with 2% FCS, we tested the ability of peripheral blood monocytes to turn to the route of the LC-DC lineage. In this system monocytes did not develop significant yeast cell phagocytosis, although mannose receptors were available. However, they became strong stimulators of mannan specific T cell proliferation. Phenotype development was analysed by flow cytometry using the monoclonal antibodies OKT6 (CD1a), IOT2 (HLA-DR), IOM2 (CD14) and the ligand Man-BSA-FITC. CD1a was the first marker which distinguished cultured monocytes from developing macrophages, obtained by addition of 8% human serum. Like cord blood Langerhans cells (CBLC) they internalized OKT6 in deep coated pits. They maintained a phenotype of monocyte derived Langerhans cells (MoLC) during eight days of in vitro culture, expressing CD1a, mannose receptors and HLA-DR and decreasing CD14, if left in their own conditioned medium. MoLC could be converted into macrophages by addition of human serum only within the first four days in vitro. Our data suggest that monocytes acquire an LC phenotype by autocrine stimulation.
Assuntos
Células de Langerhans/citologia , Lectinas Tipo C , Lectinas de Ligação a Manose , Monócitos/citologia , Receptores de Superfície Celular , Antígenos CD , Antígenos CD1 , Antígenos de Diferenciação Mielomonocítica , Diferenciação Celular , Meios de Cultura , Antígenos HLA-DR , Humanos , Técnicas In Vitro , Células de Langerhans/imunologia , Células de Langerhans/fisiologia , Receptores de Lipopolissacarídeos , Receptor de Manose , Monócitos/imunologia , Monócitos/fisiologia , Fagocitose , Fenótipo , Receptores Imunológicos/metabolismoRESUMO
Chemokine receptors are not only able to bind chemokines but, together with CD4, they serve as an entry door for the human immunodeficiency virus type 1 (HIV-1). The signalling capacity of chemokine receptors, which is of fundamental importance for chemokine-induced chemotaxis, is not used by HIV-1 to enter a target cell, nor by chemokines or chemokine-derived ligands to inhibit viral entry. In addition, an ill-defined signal triggered by chemokines can, under some circumstances, lead to an increase in HIV-1 expression. We show here that, in infected cells, exposure to SDF-1 leads to an increased expression of a X4 strain of HIV-1. A similar increase can be induced by an N-terminal peptide of SDF-1 which had previously been shown to elicit an intracellular calcium response and to inhibit the entry of X4 strains of HIV-1. We demonstrate the involvement of extracellular signal-regulated kinases (ERK) in this phenomenon. SDF-1 activates ERK-1 and ERK-2 in Jurkat cells. In HeLa cells, ERK-2 only is activated by SDF-1 or by a SDF-derived peptide. This ERK activation can be blocked by pertussis toxin and by the MEK inhibitor U0126. Most importantly, SDF-1-dependent HIV-1 expression is abolished by pretreating the cells with pertussis toxin or with U0126. The consequences of this SDF-1-induced, ERK-dependent modulation of HIV-1 expression in infected cells may have a clinical relevance for eradicating latent viruses.
Assuntos
Fármacos Anti-HIV/farmacologia , Quimiocinas CXC/farmacologia , HIV-1/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Replicação Viral/efeitos dos fármacos , Butadienos/farmacologia , Antígenos CD4/genética , Antígenos CD4/fisiologia , Cálcio/metabolismo , Ciclo Celular , Quimiocina CXCL12 , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Repetição Terminal Longa de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Células HeLa , Humanos , Células Jurkat , Nitrilas/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores CXCR4/fisiologia , Transcrição Gênica , TransfecçãoRESUMO
A panel of neutralizing murine monoclonal antibodies (MAbs) against Staphylococcus aureus alpha-toxin has been established, using formaline-inactivated alpha-toxin as an immunogen. Five independent groups of neutralizing epitopes have been identified representing five functionally important structures in the toxin molecule. Because none of the antibodies binds to overlapping decapeptides representing the toxin sequence or to bromocyanogen cleavage products of alpha-toxin, they may all bind to conformational epitopes. Nevertheless, they all bind to monomeric alpha-toxin in a Western blot. Three of the antibodies bind to the toxin monomer in an enzyme-linked immunosorbent assay (ELISA) in the presence, but not in the absence, of detergent. These epitopes are not accessible in hexameric toxin; two of them may represent the contact sites of the toxin monomers upon hexamerization and one is related to a structurally important glycine-rich central hinge region. Two different antibodies bind to monomeric toxin in an ELISA in the presence and absence of detergent and their epitopes are present more than once on oligomeric toxin; they bind strongly to hexameric toxin in a Western blot. The binding properties of the antibodies to alpha-toxin in different assay systems are summarized in an epitope model, which describes the presence of neutralizing domains in the different conformational steps required for pore formation.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Toxinas Bacterianas/imunologia , Proteínas Hemolisinas/imunologia , Animais , Ligação Competitiva , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Técnicas Imunoenzimáticas , Camundongos , Testes de NeutralizaçãoRESUMO
Bordetella pertussis secretes a calmodulin-stimulated adenylate cyclase toxin (CyaA) that is one of the major virulence factors of this organism. The toxin is able to enter various types of eukaryotic cells where, upon activation by calmodulin, it catalyzes the production of non-physiological amounts of cyclic AMP. The mechanism of toxin entry into target cells is unknown, although it has been shown that it does not involve receptor-mediated endocytosis. The adenylate cyclase toxin exhibits a very high affinity for calmodulin, and it has been proposed that the energy of calmodulin-binding to CyaA might be required for the entry of the toxin into the target cells [Oldenburg, D.J., Gross, M. K., Wong, C. S. & Storm, D. R. (1992) Biochemistry 31, 8884-8891]. In the present study, we have reexamined this issue by analyzing the cytotoxicity of various modified CyaA toxins that have altered calmodulin affinity. We show that despite their low affinity for calmodulin (at least 1000-times less than that of the wild type CyaA), these toxins were able to efficiently deliver their catalytic domain into the cytoplasm of the target cells, erythrocytes. These results demonstrate that high-affinity calmodulin binding is not required for the entry of B. pertussis adenylate cyclase into eukaryotic cells. However, the high-affinity of CyaA for calmodulin is crucial for an efficient synthesis of cAMP within the target cells.
Assuntos
Adenilil Ciclases/química , Toxinas Bacterianas/química , Bordetella pertussis/enzimologia , Bordetella pertussis/genética , Calmodulina/química , Eritrócitos/metabolismo , Adenilil Ciclases/sangue , Adenilil Ciclases/genética , Animais , Toxinas Bacterianas/sangue , Toxinas Bacterianas/genética , Catálise , AMP Cíclico/metabolismo , Eritrócitos/efeitos dos fármacos , Mutagênese Insercional , Ligação Proteica , OvinosRESUMO
Adenylate cyclase toxin (ACT), a virulence factor of Bordetella pertussis, acquires hemolytic and toxic activities after post-translational modification of the cyaA gene product, CyaA. The exact nature of this modification is unknown, but homology to the related repeat toxin alpha-hemolysin of Escherichia coli suggests that fatty acylation of a lysine residue may be involved. In the present study, we used an in vitro chemical approach to acylate unmodified, inactive adenylate cyclase protoxin by using a new water-soluble compound, acylpyrophosphate. We show that undirected transfer of lauric, myristic, or palmitic acid chains to the CyaA protoxin is able to confer both hemolytic and toxic activities to ACT. The chemically modified protoxin shows a specific requirement for Ca2+ ions for toxic activity, as does the wild type toxin. However, the toxic and hemolytic activities of chemically modified ACT are low in comparison to ACT modified in vivo, suggesting that in vitro fatty acylation of the protoxin involves random modification of nucleophilic residues present in the toxin in contrast to the in vivo modification of specific sites.
Assuntos
Bordetella pertussis/metabolismo , Ácidos Graxos/metabolismo , Hemólise , Fatores de Virulência de Bordetella/toxicidade , Acilação , Toxina Adenilato Ciclase , Adenilil Ciclases/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Eritrócitos/efeitos dos fármacos , Ácidos Graxos/química , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Ovinos , Fatores de Virulência de Bordetella/metabolismoRESUMO
CXCR4 is a G-coupled receptor for the stromal cell-derived factor (SDF-1) chemokine, and a CD4-associated human immunodeficiency virus type 1 (HIV-1) coreceptor. These functions were studied in a panel of CXCR4 mutants bearing deletions in the NH(2)-terminal extracellular domain (NT) or substitutions in the NT, the extracellular loops (ECL), or the transmembrane domains (TMs). The coreceptor activity of CXCR4 was markedly impaired by mutations of two Tyr residues in NT (Y7A/Y12A) or at a single Asp residue in ECL2 (D193A), ECL3 (D262A), or TMII (D97N). These acidic residues could engage electrostatical interactions with basic residues of the HIV-1 envelope protein gp120, known to contribute to the selectivity for CXCR4. The ability of CXCR4 mutants to bind SDF-1 and mediate cell signal was consistent with the two-site model of chemokine-receptor interaction. Site I involved in SDF-1 binding but not signaling was located in NT with particular importance of Glu(14) and/or Glu(15) and Tyr(21). Residues required for both SDF-1 binding and signaling, and thus probably part of site II, were identified in ECL2 (Asp(187)), TMII (Asp(97)), and TMVII (Glu(288)). The first residues () of NT also seem required for SDF-1 binding and signaling. A deletion in the third intracellular loop abolished signaling, probably by disrupting the coupling with G proteins. The identification of CXCR4 residues involved in the interaction with both SDF-1 and HIV-1 may account for the signaling activity of gp120 and has implications for the development of antiviral compounds.
Assuntos
Quimiocinas CXC/metabolismo , HIV-1/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Sequência de Aminoácidos , Fármacos Anti-HIV/metabolismo , Anticorpos Monoclonais/metabolismo , Ácido Aspártico/genética , Sítios de Ligação , Ligação Competitiva , Quimiocina CXCL12 , Ácido Glutâmico/genética , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Receptores CXCR4/imunologia , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Eletricidade Estática , Tirosina/genéticaRESUMO
The effect of Bordetella pertussis adenylate cyclase toxin (ACT) on platelet aggregation was investigated. This cell-invasive adenylate cyclase completely suppressed ADP (10 microM)-induced aggregation of rabbit platelets at 3 micrograms/ml and strongly suppressed thrombin (0. 2 U/ml)-induced aggregation at 10 micrograms/ml. The suppression was accompanied by marked increase in platelet intracellular cyclic AMP (cAMP) content and was diminished by the anti-ACT monoclonal antibody B7E11. A catalytically inactive point mutant of ACT did not show the suppressive effect. Since an increase of cAMP content is a known cause of platelet dysfunction, these results indicate that the observed platelet inactivation was due to the catalytic activity of ACT through increase of intracellular cAMP.
Assuntos
Toxina Adenilato Ciclase , Adenilil Ciclases/farmacologia , Bordetella pertussis/enzimologia , Toxina Pertussis , Agregação Plaquetária , Fatores de Virulência de Bordetella/farmacologia , Difosfato de Adenosina/metabolismo , Adenilil Ciclases/metabolismo , Animais , Tempo de Sangramento , AMP Cíclico/metabolismo , Camundongos , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Trombina/farmacologia , Fatores de Virulência de Bordetella/metabolismoRESUMO
A human monoclonal antibody, CB STL-1, against staphylococcal alpha-toxin has been established by hybridoma technology. It is of IgG1 subclass with lambda light chain and possesses a dissociation constant of 8 x 10(-10) mol/l. 1 mg of purified antibody neutralizes the hemolytic activity of 800 micrograms/ml alpha-toxin in an in vitro hemolysis assay using rabbit erythrocytes. The antibody does not bind to overlapping (7 residues) decapeptides spanning the sequence of alpha toxin, thus it might bind to a conformational epitope. The epitope recognized by the antibody is not accessible in oligomeric toxin. The antibody binds both to the hydrophilic and amphipathic forms of the monomeric toxin Fab fragments of the antibody are stable and show no significant loss of activity. CB STL-1 was able to protect mice in vivo from i.p. challenge with alpha toxin. Thus, the antibody is a candidate for passive immunotherapy. The variable regions of the antibody secreted by CB STL-1 were sequenced and found to be encoded by a VH gene segment belonging to the VH1 family, and a Vlambda segment most likely belonging to the VlambdaIII subgroup. Further analysis concerning the third complementarity determining region (CDR3) of the heavy chain is presented.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Toxinas Bacterianas/imunologia , Proteínas Hemolisinas/imunologia , Staphylococcus aureus/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/genética , Anticorpos Monoclonais/genética , Especificidade de Anticorpos , Toxinas Bacterianas/toxicidade , Sequência de Bases , DNA/genética , Primers do DNA/genética , Feminino , Genes de Imunoglobulinas , Proteínas Hemolisinas/toxicidade , Humanos , Hibridomas/imunologia , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Reação em Cadeia da Polimerase , CoelhosRESUMO
The CXCR-4 chemokine receptor and CD4 behave as coreceptors for cell line-adapted human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and for dual-tropic HIV strains, which also use the CCR-5 coreceptor. The cell line-adapted HIV-1 strains LAI and NDK and the dual-tropic HIV-2 strain ROD were able to infect CD4+ cells expressing human CXCR-4, while only LAI was able to infect cells expressing the rat homolog of CXCR-4. This strain selectivity was addressed by using human-rat CXCR-4 chimeras. All chimeras tested mediated LAI infection, but only those containing the third extracellular domain (e3) of human CXCR-4 mediated NDK and ROD infection. The e3 domain might be required for the functional interaction of NDK and ROD, but not LAI, with CXCR-4. Alternatively, LAI might also interact with e3 but in a different way. Monoclonal antibody 12G5, raised against human CXCR-4, did not stain cells expressing rat CXCR-4. Chimeric human-rat CXCR-4 allowed us to map the 12G5 epitope in the e3 domain. The ability of 12G5 to neutralize infection by certain HIV-1 and HIV-2 strains is also consistent with the role of e3 in the coreceptor activity of CXCR-4. The deletion of most of the amino-terminal extracellular domain (e1) abolished the coreceptor activity of human CXCR-4 for ROD and NDK but not for LAI. These results indicate that HIV strains have different requirements for their interaction with CXCR-4. They also suggest differences in the interaction of dual-tropic HIV with CCR-5 and CXCR-4.
Assuntos
HIV-1/crescimento & desenvolvimento , Proteínas de Membrana/química , Receptores de HIV/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Mapeamento de Epitopos , Espaço Extracelular , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/fisiopatologia , HIV-2/crescimento & desenvolvimento , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ratos , Receptores CXCR4 , Receptores de HIV/metabolismo , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
CCR5 and CXCR4 are the principal CD4-associated coreceptors used by human immunodeficiency virus type 1 (HIV-1). CXCR4 is also a receptor for the feline immunodeficiency virus (FIV). The rat CXCR4 cannot mediate infection by HIV-1NDK or by FIVPET (both cell line-adapted strains) because of sequence differences with human CXCR4 in the second extracellular loop (ECL2). Here we made similar observations for HIV-189.6 (a strain also using CCR5) and for a primary HIV-1 isolate. It showed the role of ECL2 in the coreceptor activity of CXCR4 for different types of HIV-1 strains. By exchanging ECL2 residues between human and rat CXCR4, we found that several amino acid differences contributed to the inactivity of the rat CXCR4 toward HIV-189.6. In contrast, its inactivity toward HIV-1NDK seemed principally due to a serine at position 193 instead of to an aspartic acid (Asp193) in human CXCR4. Likewise, a mutation of Asp187 prevented usage of CXCR4 by FIVPET. Different mutations of Asp193, including its replacement by a glutamic acid, markedly reduced or suppressed the activity of CXCR4 for HIV-1NDK infection, indicating that the negative charge was not the only requirement. Mutations of Asp193 and of arginine residues (Arg183 and Arg188) of CXCR4 reduced the efficiency of HIV-1 infection for all HIV-1 strains tested. Other ECL2 mutations tested had strain-specific effects or no apparent effect on HIV-1 infection. The ECL2 mutants allowed us to identify residues contributing to the epitope of the 12G5 monoclonal antibody. Overall, residues with different charges and interspersed in ECL2 seem to participate in the coreceptor activity of CXCR4. This suggests that a conformational rather than linear epitope of ECL2 contributes to the HIV-1 binding site. However, certain HIV-1 and FIV strains seem to require the presence of a particular ECL2 residue.
Assuntos
HIV-1/fisiologia , Vírus da Imunodeficiência Felina/fisiologia , Receptores CXCR4/genética , Replicação Viral/genética , Sequência de Aminoácidos , Animais , Gatos , Humanos , Dados de Sequência Molecular , Mutação , Conformação Proteica , Ratos , Receptores CXCR4/químicaRESUMO
Human immunodeficiency virus type 2 (HIV-2) strains that infect cells in the absence of cellular CD4 emerge spontaneously in vitro after culture in CD4+ T-cell lines. The HIV-2ROD/B strain can use the CXCR4 chemokine receptor for efficient entry into CD4+ cells. Here we have shown that the rat homologue of CXCR4, in the absence of CD4, failed to mediate CD4-independent entry by ROD/B. Furthermore, using rat-human chimeric CXCR4 receptors we have demonstrated that the second extracellular loop (E2) of human CXCR4 is critical for HIV-2 infection of CD4+ cells. E2 is also important for HIV-1 infection of CD4+ cells. Our results therefore indicate that the role of E2 in HIV entry is conserved for HIV-1 and HIV-2 and for infection in the presence or absence of CD4.
Assuntos
Antígenos CD4/metabolismo , HIV-2/metabolismo , Receptores CXCR4/metabolismo , Animais , Sítios de Ligação , Gatos , Linhagem Celular , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , HIV-1/metabolismo , HIV-1/fisiologia , HIV-2/fisiologia , Humanos , Ratos , Receptores CXCR4/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Small compounds capable of blocking the stromal cell-derived factor 1 (SDF-1) receptor CXCR4 may be potentially useful as anti-inflammatory, antiallergic, immunomodulatory, and anti-human immunodeficiency virus (HIV) agents. SDF-1-derived peptides have proven to target CXCR4 efficiently despite a 100-fold lower affinity (or more) than SDF-1. Here we studied the binding and antiviral properties of a series of substituted SDF-1-derived N-terminal peptides and tested their functional effects on human polymorphonuclear cells, because these cells are very reactive to chemokines and chemoattractants. All peptides bound to CXCR4 and inhibited HIV entry in a functional assay on CD4(+) HeLa cells. A 10-residue substituted dimer, derived from the 5-14 sequence of SDF-1, displayed the highest affinity for CXCR4 (K(i) value of 290 nM, a reduction of only 15-fold compared with SDF-1) and was also the best competitor for HIV entry (IC(50) value of 130 nM). Whereas most peptides displayed CXCR4-independent functional effects on human polymorphonuclear cells, including the modulation of calcium fluxes and the activation of superoxide anion production at high concentration (10 microM), the peptide dimer was devoid of these nonspecific effects at antiviral concentrations. Overall, this study shows that appropriate modifications of SDF-1-derived N-terminal peptides may ameliorate their binding and viral blocking properties without generating significant unspecific side effects.
Assuntos
Quimiocinas CXC/farmacologia , Neutrófilos/efeitos dos fármacos , Antivirais/farmacologia , Transporte Biológico , Cálcio/metabolismo , Quimiocina CXCL12 , Dimerização , Humanos , Técnicas In Vitro , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Fragmentos de Peptídeos/farmacologia , Receptores CXCR4/efeitos dos fármacos , Receptores CXCR4/metabolismoRESUMO
The bicyclam AMD3100 is a potent and selective inhibitor of the replication of human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). It was recently demonstrated that the compound inhibited HIV entry through CXCR4 but not through CCR5. Selectivity of AMD3100 for CXCR4 was further indicated by its lack of effect on HIV-1 and HIV-2 infection mediated by the CCR5, CCR3, Bonzo, BOB, and US28, coreceptors. AMD3100 completely blocked HIV-1 infection mediated by a mutant CXCR4 bearing a deletion of most of the amino-terminal extracellular domain. In contrast, relative resistance to AMD3100 was conferred by different single amino acid substitutions in the second extracellular loop (ECL2) or in the adjacent membrane-spanning domain, TM4. Only substitutions of a neutral residue for aspartic acid and of a nonaromatic residue for phenylalanine (Phe) were associated with drug resistance. This suggests a direct interaction of AMD3100 with these amino acids rather than indirect effects of their mutation on the CXCR4 structure. The interaction of aspartic acids of ECL2 and TM4 with AMD3100 is consistent with the positive charge of bicyclams, which might block HIV-1 entry by preventing electrostatic interactions between CXCR4 and the HIV-1 envelope protein gp120. Other features of AMD3100 must account for its high antiviral activity, in particular the presence of an aromatic linker between the cyclam units. This aromatic group might engage in hydrophobic interactions with the Phe-X-Phe motifs of ECL2 or TM4. These results confirm the importance of ECL2 for the HIV coreceptor activity of CXCR4.
Assuntos
HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Benzilaminas , Ciclamos , HIV-1/fisiologia , HIV-2/fisiologia , Células HeLa , Compostos Heterocíclicos/química , Compostos Heterocíclicos/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese , Ratos , Receptores CXCR4/genética , Células Tumorais CultivadasRESUMO
We developed a recombinant virus technique to determine the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from plasma samples, the source expected to represent the most actively replicating virus population in infected subjects. This method is not subject to selective bias associated with virus isolation in culture, a step required for conventional tropism determination procedures. The addition of a simple subcloning step allowed semiquantitative evaluation of virus populations with a different coreceptor (CCR5 or CXCR4) usage specificity present in each plasma sample. This procedure detected mixtures of CCR5- and CXCR4-exclusive virus populations as well as dualtropic viral variants, in variable proportions. Sequence analysis of dualtropic clones indicated that changes in the V3 loop are necessary for the use of CXCR4 as a coreceptor, but the overall context of the V1-V3 region is important to preserve the capacity to use CCR5. This convenient technique can greatly assist the study of virus evolution and compartmentalization in infected individuals.