RESUMEN
Through their function as epigenetic readers of the histone code, the BET family of bromodomain-containing proteins regulate expression of multiple genes of therapeutic relevance, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have profound antiproliferative and anti-inflammatory effects which translate into efficacy in oncology and inflammation models, and the first compounds have now progressed into clinical trials. The exciting biology of the BETs has led to great interest in the discovery of novel inhibitor classes. Here we describe the identification of a novel tetrahydroquinoline series through up-regulation of apolipoprotein A1 and the optimization into potent compounds active in murine models of septic shock and neuroblastoma. At the molecular level, these effects are produced by inhibition of BET bromodomains. X-ray crystallography reveals the interactions explaining the structure-activity relationships of binding. The resulting lead molecule, I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET inhibitors.
Asunto(s)
Aminoquinolinas/síntesis química , Antiinflamatorios/síntesis química , Apolipoproteína A-I/metabolismo , Benzoatos/síntesis química , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Quinolinas/síntesis química , Factores de Transcripción/antagonistas & inhibidores , Aminoquinolinas/farmacocinética , Aminoquinolinas/farmacología , Animales , Antiinflamatorios/farmacocinética , Antiinflamatorios/farmacología , Benzoatos/farmacocinética , Benzoatos/farmacología , Proteínas de Ciclo Celular , Descubrimiento de Drogas , Humanos , Ratones , Quinolinas/farmacocinética , Quinolinas/farmacología , Relación Estructura-ActividadRESUMEN
Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which are recognized by bromodomains. While the biological and structural characterization of many bromodomain containing proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and molecular characterization of potent (nM) small molecule inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophysical studies, we have discovered that the protein-protein interactions between bromodomains and acetylated histones can be antagonized by selective small molecules that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compounds bound into bromodomains of Brd2 and Brd4 elucidate the molecular interactions of binding and explain the precisely defined stereochemistry required for activity.
Asunto(s)
Apolipoproteína A-I/genética , Benzodiazepinas/metabolismo , Benzodiazepinas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Acetilación , Secuencia de Aminoácidos , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Benzodiazepinas/síntesis química , Benzodiazepinas/química , Sitios de Unión , Cristalografía por Rayos X , Descubrimiento de Drogas , Epigenómica , Células Hep G2 , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/química , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Terapia Molecular Dirigida , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estereoisomerismo , Factores de Transcripción , Regulación hacia ArribaRESUMEN
The human immunodeficiency virus, type 1 (HIV-1) entry process is triggered by interaction between the viral envelope and a seven membrane-spanning domain receptor at the cell surface, usually the CCR5 chemokine receptor. Different naturally occurring mutations in the CCR5 gene abolish receptor function, the most frequent being a 32-nucleotide deletion resulting in a truncated protein (Delta32) lacking the last three transmembrane domains (TM5-7). This mutant is retained in the endoplasmic reticulum and exerts a trans-dominant negative (TDN) effect on the wild type, preventing its exit from this compartment. This TDN effect is often considered as evidence for the oligomerization of CCR5 during transport to the cell surface. Here we use a genetic approach to define the structural determinants of the TDN effect of the Delta32 mutant. It was abolished by certain deletions and by mutations of cysteine residues preventing formation of a disulfide link between the first and second extracellular loops, suggesting that conformation of Delta32 is important for its interaction with CCR5. To circumvent this problem, we used chimeric forms of the Delta32 and wild type CCR5, consisting in substitutions with homologous domains from the mouse CCR5. All chimeric full-length receptors were expressed at the cell surface and were functional for interaction with HIV-1 or with a chemokine ligand, when assayed. The TDN effect was only observed if both the TM3 domain in CCR5 and the TM4 domain in Delta32 were from human origin, whereas the rest of the proteins could be from either origin. This suggests that the TDN effect involves some form of interaction between these transmembrane domains. Alternatively, but less likely to us, substitutions in TM4 could affect the conformation of CCR5 in the endoplasmic reticulum but not at the cell surface. However that may be, it seems that the TDN effect of the Delta32 mutant has no bearing to the issue of CCR5 dimerization and to its possible role in the processing of the receptor to the cell surface.
Asunto(s)
Genes Dominantes , Receptores CCR5/genética , Receptores CCR5/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Línea Celular , Membrana Celular/metabolismo , Dimerización , Disulfuros , Relación Dosis-Respuesta a Droga , Endocitosis , Epítopos/química , Citometría de Flujo , Eliminación de Gen , Células HeLa , Humanos , Ligandos , Ratones , Microscopía Confocal , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutagénesis , Mutación , Plásmidos/metabolismo , Pruebas de Precipitina , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Transfección , Células Tumorales CultivadasRESUMEN
The major human immunodeficiency virus type 1 (HIV-1) coreceptors are the chemokine receptors CCR5 and CXCR4. The patterns of expression of the major coreceptors and their use by HIV-1 strains largely explain viral tropism at the level of entry. However, while virus infection is dependent upon the presence of CD4 and an appropriate coreceptor, it can be influenced by a number of factors, including receptor concentration, affinity between envelope gp120 and receptors, and potentially receptor conformation. Indeed, seven-transmembrane domain receptors, such as CCR5, can exhibit conformational heterogeneity, although the significance for virus infection is uncertain. Using a panel of monoclonal antibodies (MAbs) to CXCR4, we found that CXCR4 on both primary and transformed T cells as well as on primary B cells exhibited considerable conformational heterogeneity. The conformational heterogeneity of CXCR4 explains the cell-type-dependent ability of CXCR4 antibodies to block chemotaxis to stromal cell-derived factor 1 alpha and to inhibit HIV-1 infection. In addition, the MAb most commonly used to study CXCR4 expression, 12G5, recognizes only a subpopulation of CXCR4 molecules on all primary cell types analyzed. As a result, CXCR4 concentrations on these important cell types have been underestimated to date. Finally, while the factors responsible for altering CXCR4 conformation are not known, we found that they do not involve CXCR4 glycosylation, sulfation of the N-terminal domain of CXCR4, or pertussis toxin-sensitive G-protein coupling. The fact that this important HIV-1 coreceptor exists in multiple conformations could have implications for viral entry and for the development of receptor antagonists.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1/inmunología , Receptores CXCR4/inmunología , Secuencia de Aminoácidos , Epítopos/química , Epítopos/inmunología , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Receptores CXCR4/químicaRESUMEN
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.
Asunto(s)
Quimiocinas CXC/farmacología , Neutrófilos/efectos de los fármacos , Antivirales/farmacología , Transporte Biológico , Calcio/metabolismo , Quimiocina CXCL12 , Dimerización , Humanos , Técnicas In Vitro , Neutrófilos/metabolismo , Neutrófilos/fisiología , Fragmentos de Péptidos/farmacología , Receptores CXCR4/efectos de los fármacos , Receptores CXCR4/metabolismoRESUMEN
Human immunodeficiency virus type 1 (HIV-1) entry is triggered by the interaction of the gp120 envelope glycoprotein with a cellular chemokine receptor, either CCR5 or CXCR4. We have identified different mutations in human CXCR4 that prevent efficient infection by one HIV-1 strain (NDK) but not another (LAI) and sought to define these strain-dependent effects at the gp120 level. The lack of activity toward the NDK strain of the HHRH chimeric CXCR4 in which the second extracellular loop (ECL2) derived from the rat CXCR4 and of CXCR4 with mutations at an aspartic acid in ECL2 (D193A and D193R) was apparently due to the sequence of the third variable loop (V3) of gp120, more precisely, to its C-terminal part. Indeed, substitution of the LAI V3 loop or only its C-terminal part in the NDK gp 120 context was sufficient to restore usage of the HHRH, D193A, and D193R receptors. The same result was achieved upon mutation of a single lysine residue of the NDK V3 loop to alanine (K319A) but not to arginine (K319R). These results provide a strong case for a direct interaction between the gp120 V3 loop and the ECL2 domain of CXCR4. By contrast, V3 substitutions had no effect on the inability of NDK to infect cells via a mutant CXCR4 in which the amino-terminal extracellular domain (NT) is deleted. In experiments with a set of chimeric NDK-LAI gp120s, the V1/V2 region from LAI gp120 was both necessary and sufficient for usage of the NT-deleted CXCR4. Different variable domains of gp120 can therefore cooperate for a functional interaction with CXCR4.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/patogenicidad , Fragmentos de Péptidos/metabolismo , Receptores CXCR4/metabolismo , Secuencia de Aminoácidos , Animales , Astrocitoma , Antígenos CD4/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , Humanos , Fusión de Membrana , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Ratas , Receptores CXCR4/genética , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/virología , VIH-1/fisiología , Receptores CCR5/análisis , Receptores CXCR4/análisis , Viremia/virología , Secuencia de Aminoácidos , Células Cultivadas , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Fenotipo , Recombinación Genética , Replicación ViralAsunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/fisiología , Fusión de Membrana/efectos de los fármacos , Receptores del VIH/efectos de los fármacos , Amidas/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Humanos , Compuestos de Amonio Cuaternario/antagonistas & inhibidores , Receptores del VIH/metabolismo , Proteínas del Envoltorio Viral/metabolismoRESUMEN
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.
Asunto(s)
Quimiocinas CXC/metabolismo , VIH-1/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Secuencia de Aminoácidos , Fármacos Anti-VIH/metabolismo , Anticuerpos Monoclonales/metabolismo , Ácido Aspártico/genética , Sitios de Unión , Unión Competitiva , Quimiocina CXCL12 , Ácido Glutámico/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Receptores CXCR4/inmunología , Proteínas Recombinantes/metabolismo , Transducción de Señal , Electricidad Estática , Tirosina/genéticaRESUMEN
The triterpene RPR103611 is an efficient inhibitor of membrane fusion mediated by the envelope proteins (Env, gp120-gp41) of CXCR4-dependent (X4) human immunodeficiency virus type 1 (HIV-1) strains, such as HIV-1(LAI) (LAI). Other X4 strains, such as HIV-1(NDK) (NDK), and CCR5-dependent (R5) HIV-1 strains, such as HIV-1(ADA) (ADA), were totally resistant to RPR103611. Analysis of chimeric LAI-NDK Env proteins identified a fragment of the NDK gp41 ectodomain determining drug resistance. A single difference at position 91, leucine in LAI and histidine in NDK, apparently accounted for their sensitivity or resistance to RPR103611. We had previously identified a mutation of isoleucine 84 to serine in a drug escape LAI variant. Both I84 and L91 are located in the "loop region" of gp41 separating the proximal and distal helix domains. Nonpolar residues in this region therefore appear to be important for the antiviral activity of RPR103611 and are possibly part of its target. However, another mechanism had to be envisaged to explain the drug resistance of ADA, since its gp41 loop region was almost identical to that of LAI. Fusion mediated by chimeric Env consisting of LAI gp120 and ADA gp41, or the reciprocal construct, was fully blocked by RPR103611. The gp120-gp41 complex of R5 strains is stable, relative to that of X4 strains, and this stability could play a role in their drug resistance. Indeed, when the postbinding steps of ADA infection were performed under mildly acidic conditions (pH 6.5 or 6.0), a treatment expected to favor dissociation of gp120, we achieved almost complete neutralization by RPR103611. The drug resistance of NDK was partially overcome by preincubating virus with soluble CD4, a gp120 ligand inducing conformational changes in the Env complex. The antiviral efficacy of RPR103611 therefore depends on the sequence of the gp41 loop and the stability of the gp120-gp41 complex, which could limit the accessibility of this target.
Asunto(s)
Antivirales/farmacología , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/efectos de los fármacos , Triterpenos/farmacología , Secuencia de Aminoácidos , Línea Celular , Técnicas de Cocultivo , Farmacorresistencia Microbiana , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/química , Células HeLa , Histidina/metabolismo , Humanos , Leucina/metabolismo , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Strains of the feline immunodeficiency virus (FIV) presently under investigation exhibit distinct patterns of in vitro tropism. In particular, the adaptation of FIV for propagation in Crandell feline kidney (CrFK) cells results in the selection of strains capable of forming syncytia with cell lines of diverse species origin. The infection of CrFK cells by CrFK-adapted strains appears to require the chemokine receptor CXCR4 and is inhibited by its natural ligand, stromal cell-derived factor 1alpha (SDF-1alpha). Here we found that inhibitors of CXCR4-mediated infection by human immunodeficiency virus type I (HIV-1), such as the bicyclam AMD3100 and short peptides derived from the amino-terminal region of SDF-1alpha, also blocked infection of CrFK by FIV. Nevertheless, we observed differences in the ranking order of the peptides as inhibitors of FIV and HIV-1 and showed that such differences are related to the species origin of CXCR4 and not that of the viral envelope. These results suggest that, although the envelope glycoproteins of FIV and HIV-1 are substantially divergent, FIV and HIV-1 interact with CXCR4 in a highly similar manner. We have also addressed the role of CXCR4 in the life cycle of primary isolates of FIV. Various CXCR4 ligands inhibited infection of feline peripheral blood mononuclear cells (PBMC) by primary FIV isolates in a concentration-dependent manner. These ligands also blocked the viral transduction of feline PBMC by pseudotyped viral particles when infection was mediated by the envelope glycoprotein of a primary FIV isolate but not by the G protein of vesicular stomatitis virus, indicating that they act at an envelope-mediated step and presumably at viral entry. These findings strongly suggest that primary and CrFK-adapted strains of FIV, despite disparate in vitro tropisms, share usage of CXCR4.
Asunto(s)
Virus de la Inmunodeficiencia Felina/metabolismo , Receptores CXCR4/metabolismo , Adaptación Biológica , Animales , Fármacos Anti-VIH/farmacología , Gatos , Línea Celular Transformada , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Concanavalina A/farmacología , Células HeLa , Humanos , Virus de la Inmunodeficiencia Felina/efectos de los fármacos , Virus de la Inmunodeficiencia Felina/aislamiento & purificación , Virus de la Inmunodeficiencia Felina/fisiología , Ligandos , Activación de Linfocitos , Linfocitos/virología , Ratones , Mitógenos/farmacologíaRESUMEN
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.
Asunto(s)
VIH-1/fisiología , Virus de la Inmunodeficiencia Felina/fisiología , Receptores CXCR4/genética , Replicación Viral/genética , Secuencia de Aminoácidos , Animales , Gatos , Humanos , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Ratas , Receptores CXCR4/químicaRESUMEN
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.
Asunto(s)
VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Receptores CXCR4/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Bencilaminas , Ciclamas , VIH-1/fisiología , VIH-2/fisiología , Células HeLa , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/metabolismo , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis , Ratas , Receptores CXCR4/genética , Células Tumorales CultivadasRESUMEN
The human cytomegalovirus (CMV) US28 gene encodes a functional CC chemokine receptor. However, this activity was observed in cells transfected to express US28 and might not correspond to the actual role of the protein in the CMV life cycle. Expression of US28 allows human immunodeficiency virus type 1 (HIV-1) entry into certain CD4(+) cells and their fusion with cells expressing HIV-1 envelope (Env) proteins. Such properties were initially reported for the cellular chemokine receptors CCR5 and CXCR4, which behave as CD4-associated HIV-1 coreceptors. We found that coexpression of US28 and either CXCR4 or CCR5 in CD4(+) cells resulted in enhanced synctium formation with HIV-1 Env+ cells. This positive effect of US28 on cell fusion seems to be distinct from its HIV-1 coreceptor activity. Indeed, enhancement of cell fusion was also observed when US28 was expressed on the HIV-1 Env+ cells instead of an CD4(+) target cells. Furthermore, US28 could enhance cell fusion mediated by other viral proteins, in particular, the G protein of vesicular stomatitis virus (VSV-G). The HIV-1 coreceptor and fusion-enhancing activities could be affected by mutations in different domains of US28. The fusion-enhancing activity of US28 seems to be cell type dependent. Indeed, cells coexpressing VSV-G and US28 fused more efficiently with human, simian, or feline target cells, while US28 had no apparent effect on fusion with the three mouse or rat cell lines tested. The positive effect of US28 on cell fusion might therefore require its interaction with a cell-specific factor. We discuss a possible role for US28 in the fusion of the CMV envelope with target cells and CMV entry.
Asunto(s)
Citomegalovirus/metabolismo , Fusión de Membrana , Glicoproteínas de Membrana , Receptores de Quimiocina/metabolismo , Proteínas Virales/metabolismo , Células 3T3 , Animales , Gatos , Línea Celular , Productos del Gen env/genética , Productos del Gen env/metabolismo , Células HeLa , Humanos , Macaca mulatta , Ratones , Mutagénesis , Ratas , Receptores CCR2 , Receptores de Quimiocina/genética , Proteínas Oncogénicas de Retroviridae/genética , Proteínas Oncogénicas de Retroviridae/metabolismo , Células Tumorales Cultivadas , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/genética , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
The feline homolog of the alpha-chemokine receptor CXCR4 has recently been shown to support cell-cell fusion mediated by CXCR4-dependent strains of human immunodeficiency virus (HIV) and strains of feline immunodeficiency virus (FIV) that have been selected for growth in the Crandell feline kidney (CrFK) cell line. In this report we demonstrate that expression of CXCR4 alone is sufficient to render cells from diverse species permissive for fusion with FIV-infected cells, suggesting that CXCR4 is the sole receptor for CrFK-tropic strains of FIV, analogous to CD4-independent strains of HIV-2. To identify the regions of CXCR4 involved in fusion mediated by FIV, we screened panels of chimeric CXCR4 molecules for the ability to support fusion with FIV-infected cells. Human CXCR4 supported fusion more efficiently than feline CXCR4 and feline/human CXCR4 chimeras, suggesting that the second and third extracellular loops of human CXCR4 contain a critical determinant for receptor function. Rat/human CXCR4 chimeras suggested that the second extracellular loop contained the principal determinant for receptor function; however, chimeras constructed between human CXCR2 and CXCR4 revealed that the first and third loops of CXCR4 contribute to the FIV Env binding site, as replacement of these domains with the corresponding domains of CXCR2 rendered the molecule nonfunctional in fusion assays. Mutation of the DRY motif and the C-terminal cytoplasmic tail of CXCR4 did not affect the ability of the molecule to support fusion, suggesting that neither signalling via G proteins nor receptor internalization was required for fusion mediated by FIV; similarly, truncation of the N terminus of CXCR4 did not affect the function of the molecule as a receptor for FIV. CXCR4-transfected feline cells were rendered permissive for infection with both the CrFK-tropic PET isolate of FIV and the CXCR4-dependent RF strain of HIV-1, and susceptibility to infection correlated well with ability to support fusion. The data suggest that the second extracellular loop of CXCR4 is the major determinant of CXCR4 usage by FIV.
Asunto(s)
Virus de la Inmunodeficiencia Felina/metabolismo , Receptores CXCR4/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Células CHO , Gatos , Cricetinae , ADN Complementario , Humanos , Virus de la Inmunodeficiencia Felina/fisiología , Fusión de Membrana , Ratones , Datos de Secuencia Molecular , Ratas , Receptores CXCR4/genética , Transfección , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Antígenos CD4/metabolismo , VIH-2/metabolismo , Receptores CXCR4/metabolismo , Animales , Sitios de Unión , Gatos , Línea Celular , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , VIH-1/metabolismo , VIH-1/fisiología , VIH-2/fisiología , Humanos , Ratas , Receptores CXCR4/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
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.
Asunto(s)
Fármacos Anti-VIH/farmacología , Quimiocinas CXC/farmacología , VIH-1/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Fármacos Anti-VIH/química , Quimiocina CXCL12 , Quimiocinas CXC/química , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Infecciones por VIH/prevención & control , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Receptores CXCR4/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The eotaxin receptor (CCR3) is a CD4-associated coreceptor for human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). By comparison with other chemokine receptors, such as CCR5 and CXCR4, the primary sequences of human CCR3 and its rhesus macaque homolog were markedly different in their extracellular domains. Human CD4+ cells expressing CCR3 from either human or macaque origin could be infected by HIV-2, with apparently similar efficiency, but only cells expressing human CCR3 could be infected by HIV-1. It suggests that HIV-1 and HIV-2 envelope proteins interact differently with the CCR3 coreceptor HIV-1 could infect cells expressing chimeric human/macaque CCR3 bearing either the first and second, or the third and fourth extracellular domains of human CCR3. As previously observed for CCR5, there seems to be a certain functional redundancy between domains supporting the coreceptor activity of CCR3. In spite of their close genetic relationship to HIV-2, two macaque simian immunodeficiency virus strains were apparently unable to use the CCR3 coreceptor from either human or simian origin.
Asunto(s)
VIH-1/fisiología , VIH-2/fisiología , Receptores de Quimiocina/fisiología , Secuencia de Aminoácidos , Animales , Linfocitos T CD4-Positivos , Línea Celular , Quimera , Chlorocebus aethiops , Células HeLa , Humanos , Macaca mulatta , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores CCR3 , Receptores de Quimiocina/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Especificidad por SustratoRESUMEN
A triterpene derived from betulinic acid (RPR103611) blocks human immunodeficiency virus type 1 (HIV-1) infection and fusion of CD4+ cells with cells expressing HIV-1 envelope proteins (gp120 and gp41), suggesting an effect on virus entry. This compound did not block infection by a subtype D HIV-1 strain (NDK) or cell-cell fusion mediated by the NDK envelope proteins. The genetic basis of drug resistance was therefore addressed by testing envelope chimeras derived from NDK and a drug-sensitive HIV-1 strain (LAI, subtype B). A drug-resistant phenotype was observed for all chimeras bearing the ectodomain of NDK gp41, while the origins of gp120 and of the membrane anchor and cytoplasmic domains of gp41 had no apparent role. The envelope gene of a LAI variant, fully resistant to the antiviral effect of RPR103611, was cloned and sequenced. Its product differed from the parental sequence at two positions in gp41, with changes of arginine 22 to alanine (R22A) and isoleucine 84 to serine (I84S), the gp120 being identical. In the context of LAI gp41, the I84S substitution was sufficient for drug resistance. Therefore, in two different systems, differences in gp41 were associated with sensitivity or resistance to RPR103611. Modifications of gp41 can affect the quaternary structure of gp120 and gp41 and the accessibility of gp120 to antiviral agents such as neutralizing antibodies. However, a direct effect of RPR103611 on a gp41 target must also be envisioned, in agreement with the blocking of apparently late steps of HIV-1 entry. This compound could be a valuable tool for structure-function studies of gp41.