RESUMO
The chemokine receptor CXCR4 is the major coreceptor used for cellular entry by T cell- tropic human immunodeficiency virus (HIV)-1 strains, whereas CCR5 is used by macrophage (M)-tropic strains. Here we show that a small-molecule inhibitor, ALX40-4C, inhibits HIV-1 envelope (Env)-mediated membrane fusion and viral entry directly at the level of coreceptor use. ALX40-4C inhibited HIV-1 use of the coreceptor CXCR4 by T- and dual-tropic HIV-1 strains, whereas use of CCR5 by M- and dual-tropic strains was not inhibited. Dual-tropic viruses capable of using both CXCR4 and CCR5 were inhibited by ALX40-4C only when cells expressed CXCR4 alone. ALX40-4C blocked stromal-derived factor (SDF)-1alpha-mediated activation of CXCR4 and binding of the monoclonal antibody 12G5 to cells expressing CXCR4. Overlap of the ALX40-4C binding site with that of 12G5 and SDF implicates direct blocking of Env interactions, rather than downregulation of receptor, as the mechanism of inhibition. Thus, ALX40-4C represents a small-molecule inhibitor of HIV-1 infection that acts directly against a chemokine receptor at the level of Env-mediated membrane fusion.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , Oligopeptídeos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Humanos , Linfoma de Células T , Oligopeptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptores CXCR4/metabolismo , Linfócitos T/virologia , Células Tumorais CultivadasRESUMO
Retroviral vectors containing CD4 and an appropriate chemokine receptor were evaluated for the ability to transduce cells infected with human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). These CD4-chemokine receptor pseudotypes were able to target HIV- and SIV-infected cell lines and monocyte-derived macrophages in a manner that corresponded to the specificity of the viral envelope glycoprotein for its CD4-chemokine receptor complex. This approach could offer a way to deliver antiviral genes directly to HIV-infected cells in vivo and could provide an additional treatment strategy in conjunction with existing antiviral therapies.
Assuntos
Antígenos CD4/genética , Técnicas de Transferência de Genes , Vetores Genéticos , HIV-1/fisiologia , Receptores de Quimiocinas/genética , Vírus da Imunodeficiência Símia/fisiologia , Animais , Antígenos CD4/metabolismo , Linhagem Celular , Produtos do Gene env/metabolismo , Humanos , Macrófagos/virologia , Plasmídeos , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores de Quimiocinas/metabolismo , TransfecçãoRESUMO
Intracellular dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed to alter endosomal acidification. The most widely studied consequence of this defect has been alterations in the biochemical properties of cystic fibrosis (CF) respiratory mucus glycoproteins. However, studies confirming the existence of mucous processing defects in CF have been hindered by the lack of in vivo animal models by which to test these hypotheses in the absence of secondary effects of chronic bacterial infection. The human bronchial xenograft model has been useful in evaluating the pathophysiologic differences between CF and non-CF airway epithelium, in the absence of secondary disease effects such as goblet cell hyperplasia. In this study we sought to compare the extent of sulfation within secreted mucus glycoproteins from CF and non-CF human bronchial xenografts. Cumulative results of xenografts generated from 13 independent CF tissue samples demonstrated a statistically significant higher level of sulfation (1.7 +/- 0.18, P < 0.026) as compared to non-CF paired controls. Such findings add to the growing body of knowledge that primary defects in sulfation exist in CF respiratory mucin. Correlation of genotype with the extent of mucus sulfation revealed two categories of CF tissues with statistically different mucus sulfation profiles. Results from these studies demonstrated a 2.0 +/- 0.15-fold higher level of mucus sulfation produced from xenografts of five defined CF genotypes as compared to non-CF controls (P < 0.004, n= 10). Interestingly, three CF samples for which one mutant allele remained undefined (deltaoff8/unknown or G551D/unknown) demonstrated no statistical difference in the level of sulfation as compared with matched non-CF controls (n= 3). This as yet unknown allele was not identified within a screen for the 26 most common CF mutations. These results provide preliminary evidence for allelic variation within the CF population which may begin to elucidate the structure-function of CFTR with regards to intracellular mucus processing defects.
Assuntos
Brônquios/metabolismo , Fibrose Cística/genética , Mucinas/biossíntese , Mucinas/genética , Mutação , Adolescente , Adulto , Animais , Brônquios/transplante , Células Cultivadas , Fibrose Cística/metabolismo , Fibrose Cística/cirurgia , Feminino , Glucosamina/metabolismo , Humanos , Transplante de Pulmão , Masculino , Camundongos , Camundongos Nus , Mucinas/isolamento & purificação , Mutação Puntual , Técnica de Diluição de Radioisótopos , Valores de Referência , Sulfatos/metabolismo , Radioisótopos de Enxofre , Transplante Heterólogo , TrítioRESUMO
We have evaluated the biological efficacy of E1-deleted adenoviruses in baboons for lung-directed gene therapy of cystic fibrosis (CF). The experimental design attempted to simulate a phase I clinical trial with animals receiving a single dose of virus to an isolated pulmonary segment. A total of 14 animals divided into four groups, each of which received escalating doses of virus, were used. Individual animals were necropsied 4 and 21 days after gene transfer and tissues were carefully surveyed for gene expression. Expression of the transgene was localized primarily to the area into which it was infused; the efficiency of recombinant gene expression and the abundance of transgene sequences were proportional to dose and both diminished with time. Transgene expression was found predominantly in alveolar cells with patches of expression in the proximal and distal airway. Analysis of adenoviral protein expression within transgene-expressing cells revealed infrequent expression of the E2a gene and no detectable expression of late genes (i.e., fiber protein). These results suggest that recombinant adenovirus can be used to transfer genes efficiently to the lung of nonhuman primates and that therapeutic strategies of cystic fibrosis may require repetitive administration with current vectors.
Assuntos
Adenoviridae/genética , Fibrose Cística/terapia , Terapia Genética , Pulmão/metabolismo , Proteínas de Membrana/genética , Adenoviridae/isolamento & purificação , Animais , Regulador de Condutância Transmembrana em Fibrose Cística , DNA Viral/análise , Estudos de Avaliação como Assunto , Expressão Gênica , Vetores Genéticos , Imuno-Histoquímica , Masculino , Proteínas de Membrana/uso terapêutico , Papio , Distribuição TecidualRESUMO
CD4 is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1), but is not sufficient for entry of HIV-1 into cells. After a decade-long search, the cellular coreceptors that HIV-1 requires in conjunction with CD4 have been identified as members of the chemokine receptor family of seven-transmembrane G-protein coupled receptors. The discovery of distinct chemokine receptors that support entry of T-cell tropic (CXCR-4) and macrophage tropic HIV-1 strains (CCR-5) explains the differences in cell tropism between viral strains, the inability of HIV-1 to infect most nonprimate cells, and the resistance of a small percentage of the population to HIV-1 infection. Further understanding of the role of chemokine receptors in viral entry may also help explain the evolution of more pathogenic forms of the virus, viral transmission, and HIV-induced pathogenesis. These recent discoveries will aid the development of strategies for combating HIV-1 transmission and spread, the understanding of HIV-1 fusion mechanisms, and the possible development of small animal models for HIV-1 drug and vaccine testing.
Assuntos
Quimiocinas/fisiologia , HIV-1/imunologia , Fusão de Membrana , Receptores de Citocinas/fisiologia , Receptores de HIV/fisiologia , Antígenos CD4 , Quimiocinas/imunologia , HIV-1/patogenicidade , Humanos , Proteínas de Membrana , Polimorfismo Genético/imunologia , Receptores CCR5 , Receptores CXCR4RESUMO
ALX40-4C is a small peptide inhibitor of the chemokine receptor CXCR4 that can inhibit X4 strains of HIV-1. Prior to the discovery of chemokine receptors as the HIV coreceptors, ALX40-4C was used in phase I/II clinical trials to evaluate its therapeutic potential against HIV-1, making ALX40-4C the first anticoreceptor inhibitor to be tested in humans against HIV-1. Patients in the highest dose groups achieved ALX40-4C levels above the effective concentration of the drug for nearly the entire 1-month treatment period. ALX40-4C was well tolerated by 39 of 40 asymptomatic HIV-infected patients, despite the critical role of CXCR4 in normal development and hematopoiesis. No significant or consistent reductions in viral load were observed, but only 12 of the enrolled patients harbored virus types that used CXCR4. We also found that ALX40-4C interacts with the second extracellular loop of CXCR4 and inhibits infection exclusively by blocking direct virus-CXCR4 interactions.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Oligopeptídeos/uso terapêutico , Receptores CXCR4/antagonistas & inibidores , Adulto , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacocinética , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular , Qualidade de Produtos para o Consumidor , Feminino , Infecções por HIV/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Oligopeptídeos/administração & dosagem , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacocinética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores CXCR4/fisiologiaAssuntos
Antígenos CD4/metabolismo , Fusão Celular , HIV-1/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores de HIV/metabolismo , Animais , Linhagem Celular , RNA Polimerases Dirigidas por DNA/genética , Expressão Gênica , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Genes Reporter , Humanos , Imuno-Histoquímica , Cinética , Fusão de Membrana , Codorniz , Receptores de Quimiocinas/análise , Transfecção , Vaccinia virus , Proteínas ViraisRESUMO
Binding of the extracellular subunit of human immunodeficiency type 1 (HIV-1) envelope (Env) glycoprotein (gp120) to CD4 triggers the induction or exposure of a highly conserved coreceptor binding site in gp120 that helps mediate membrane fusion. Characterizing the structural features involved in gp120-coreceptor binding and the conditions under which binding occurs is important for understanding the fusion process, the evolution of pathogenic strains in vivo, the identification of novel anti-HIV compounds, and the development of HIV vaccines that utilize triggered structures of Env. Here we use the kinetics of interaction between CCR5 and gp120 to understand temporal and structural changes that occur during viral fusion. Using saturation binding and homologous competition analysis, we estimated the K(d) of interaction between CCR5 and gp120 from the macrophage tropic HIV-1 strain JRFL to be 4 nM. Unlike Env-mediated fusion, gp120 binding to CCR5 did not require divalent cations or elevated temperatures. Binding was not significantly affected by the pH of binding, G-protein coupling of CCR5, or partial gp120 deglycosylation. Oligomeric, uncleaved JRFL gp140 failed to bind CCR5 despite its ability to bind CD4 and monoclonal antibody 17b, suggesting that the uncleaved ectodomain of gp41 interferes with full exposure of the chemokine receptor binding site. Exposure of the chemokine receptor binding site on gp120 could be induced rapidly by CD4, but exposure of this site was lost upon CD4 dissociation from gp120, indicating that the conformational changes in gp120 induced by CD4 binding are fully reversible. The functional gp120-soluble CD4 complex was remarkably stable over time and temperature ranges, offering the possibility that complexes in which the highly conserved coreceptor binding site in gp120 is exposed can be used for vaccine development.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Fusão de Membrana/fisiologia , Receptores CCR5/metabolismo , Animais , Antígenos CD4/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Chlorocebus aethiops , Cricetinae , Meios de Cultura , Produtos do Gene env/metabolismo , Glicosilação , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Células HeLa , Humanos , Cinética , Oligopeptídeos/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Temperatura , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
Interaction of HIV and SIV Envelope (Env) proteins with viral coreceptors is a critical step in viral entry. By using a sensitive and specific gp120 binding assay, we have identified a discordance between the ability of a coreceptor to support Env-mediated membrane fusion and high-affinity binding of gp120. Direct binding of gp120 from the dual-tropic HIV-1 strain 89.6 was not detectable for any coreceptor that it uses for fusion, while detectable binding of gp120s from the R5 HIV-1 strains JRFL and CM235 and the SIV strain 239 was not measurable for many CCR5 chimeras and mutants that function efficiently as viral coreceptors. In comparison, binding of chemokines to these same mutants was highly predictive of their ability to signal. Thus, gp120 is more sensitive than chemokines to perturbations of CCR5 structure. We conclude that while chemokine binding to CCR5 is a good predictor of chemokine receptor function, gp120 binding does not always predict coreceptor function.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Glicoproteínas de Membrana , Receptores CCR5/metabolismo , Receptores de HIV/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Proteínas do Envelope Viral , Antígenos CD4/metabolismo , Linhagem Celular , Infecções por HIV/virologia , Humanos , Fusão de Membrana , Receptores CCR5/química , Receptores de Quimiocinas/metabolismo , Transdução de SinaisRESUMO
First-generation recombinant adenoviruses that lack E1 sequences have shown tremendous promise in animal and human models of gene therapy. Important limitations of these vectors are that recombinant gene expression is transient and inflammation occurs at the site of gene transfer. Our hypothesis for generating vectors with increased persistence is that present recombinant adenoviruses express viral proteins that stimulate cellular immune responses leading to destruction of the infected cells and repopulation of the organ with non-transgene-containing cells. This model predicts that further crippling of the virus will improve persistence and diminish pathology. We describe in this report second-generation recombinant adenoviruses harboring a beta-galactosidase-expressing transgene in which a temperature-sensitive mutation has been introduced into the E2A gene of an E1-deleted recombinant. At nonpermissive temperature, this virus fails to express late gene products, even when E1 is expressed in trans. The biology of this recombinant was studied in vivo in the context of mouse liver, a setting that is permissive for adenovirus type 5 replication. Animals that received the second-generation virus expressed the transgene for at least 70 days, whereas expression of the first-generation virus was no longer than 14 days. In addition, the inflammatory response, as measured by infiltration of CD8+ T cells, was blunted and delayed in livers infected with second-generation virus. These studies illustrate that modifications that disrupt structural protein expression in recombinant adenoviruses may be useful in enhancing their utility for gene therapy.
Assuntos
Adenoviridae/genética , Proteínas E2 de Adenovirus/genética , Genes Virais , Inflamação/prevenção & controle , Fígado/metabolismo , Proteínas E2 de Adenovirus/biossíntese , Animais , Antígenos CD8/análise , Ciclosporina/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Deleção de Genes , Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Vetores Genéticos , Humanos , Imuno-Histoquímica , Fígado/fisiopatologia , Camundongos , Camundongos Transgênicos , beta-Galactosidase/análise , beta-Galactosidase/biossínteseRESUMO
Cellular infection by the human immunodeficiency virus type 1 (HIV-1) requires interaction of the viral envelope protein with CD4 and at least one additional cell surface molecule, termed a "cofactor" or "coreceptor." Recent discoveries have determined that macrophage-tropic strains of HIV-1 which are largely responsible for sexual transmission require the beta-chemokine receptor CCR5 in addition to CD4, while the T cell tropic viruses that emerge later after infection use the alpha-chemokine receptor CXCR4. Thus, both CD4 and the appropriate chemokine receptor must be expressed on the cell surface in order for HIV-1 to enter the cell and establish an infection. The in vivo importance of CCR5 for HIV-1 is demonstrated by the finding that individuals homozygous for a 32 bp deletion (delta 32) in the CCR5 gene that renders them effectively CCR5-negative are highly resistant to virus infection. In this review, the structure-function correlates of the chemokine receptors that serve as major coreceptors for HIV-1 and simian immunodeficiency virus entry will be reviewed. Since certain chemokines have been implicated as stem cell inhibitory factors, the biological consequences of chemokine receptor expression as it relates to HIV-1-associated hematodyspoiesis will also be discussed.
Assuntos
HIV-1/metabolismo , Hematopoese , Receptores de Quimiocinas/metabolismo , Animais , Humanos , Polimorfismo Genético , Receptores de Quimiocinas/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Entry of human immunodeficiency virus type 1 (HIV-1) into cells requires binding to CD4 and fusion with a cellular membrane. Fusion does not occur in most nonhuman cells even when they express human CD4, indicating that one or more human accessory factors are required for virus infection. Recently, a seven-transmembrane domain protein has been shown to serve as an accessory factor for T-cell-tropic (T-tropic) HIV-1 isolates (Y. Feng, C. C. Broder, P. E. Kennedy, and E. A. Berger, Science 272:872-877, 1996). Here we show that expression of this glycoprotein, termed fusin, in murine, feline, simian, and quail cell lines, in conjunction with human CD4, rendered these cells fully permissive for HIV-1 envelope glycoprotein (Env)-mediated membrane fusion. Expression of CD4 or fusin alone did not permit fusion. In addition, introduction of fusin and CD4 into a human cell line, U87MG, that is resistant to HIV-1 induced syncytium formation and to infection by HIV-1 when expressing CD4 alone made this cell line permissive for Env-mediated cell-cell fusion. Fusion was observed only with T-tropic Env proteins. Macrophage-tropic (M-tropic) Env proteins from the SF162, ADA, and Ba-L HIV-1 strains did not fuse with cells expressing fusin and CD4, suggesting that M-tropic viruses utilize an accessory molecule other than fusin. Finally, coexpression of fusin and CD4 made both a murine and feline cell line susceptible to virus infection by T-tropic, but not M-tropic, HIV-1 strains.
Assuntos
Antígenos CD4/fisiologia , HIV-1/fisiologia , Proteínas de Membrana/fisiologia , Receptores de HIV/fisiologia , Linfócitos T/imunologia , Linfócitos T/virologia , Sequência de Aminoácidos , Animais , Antígenos CD/fisiologia , Astrocitoma , Sequência de Bases , Gatos , Linhagem Celular , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Clonagem Molecular , Coturnix , Primers do DNA , Feto , Biblioteca Gênica , Produtos do Gene env/fisiologia , Células Gigantes , HIV-1/imunologia , Células HeLa , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Receptores CXCR4 , Baço , Células Tumorais CultivadasRESUMO
Here, we show that the beta-chemokine receptor CKR-5 serves as a cofactor for M-tropic HIV viruses. Expression of CKR-5 with CD4 enables nonpermissive cells to form syncytia with cells expressing M-tropic, but not T-tropic, HIV-1 env proteins. Expression of CKR-5 and CD4 enables entry of a M-tropic, but not a T-tropic, virus strain. A dual-tropic primary HIV-1 isolate (89.6) utilizes both Fusin and CKR-5 as entry cofactors. Cells expressing the 89.6 env protein form syncytia with QT6 cells expressing CD4 and either Fusin or CKR-5. The beta-chemokine receptors CKR-3 and CKR-2b support HIV-1 89.6 env-mediated syncytia formation but do not support fusion by any of the T-tropic or M-tropic strains tested. Our results suggest that the T-tropic viruses characteristic of disease progression may evolve from purely M-tropic viruses prevalent early in virus infection through changes in the env protein that enable the virus to use multiple entry cofactors.
Assuntos
Fusão Celular/fisiologia , HIV-1/fisiologia , Proteínas de Membrana/fisiologia , Receptores de Quimiocinas , Receptores de Citocinas/fisiologia , Receptores de HIV/fisiologia , Síndrome da Imunodeficiência Adquirida/virologia , Sequência de Bases , Quimiocinas/fisiologia , Células HeLa/fisiologia , Células HeLa/virologia , Humanos , Dados de Sequência Molecular , Fenótipo , Receptores CCR3 , Receptores CCR5 , Receptores CXCR4 , Proteínas do Envelope Viral/genéticaRESUMO
CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for maintaining the structural integrity necessary for ligand binding and receptor activation. A natural mutation affecting Cys20, which is thought to form a disulfide bond with Cys269, has been described in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties, and its ability to function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression levels by 40-70%. However, mutation of Cys101 or Cys178, predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5 antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with partial restoration of receptor expression and conformation achieved by incubating cells at 32 degrees C. All cysteine mutants were unable to bind detectable levels of MIP-1beta, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling. Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation.
Assuntos
Quimiocinas/metabolismo , Cisteína/metabolismo , HIV-1/metabolismo , Receptores CCR5/metabolismo , Substituição de Aminoácidos , Animais , Células CHO , Linhagem Celular , Quimiocina CCL4 , Cricetinae , Dissulfetos/metabolismo , Humanos , Ligantes , Proteínas Inflamatórias de Macrófagos/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Receptores CCR5/genéticaRESUMO
CCR5 was first characterized as a receptor for MIP-1alpha, MIP-1beta, and RANTES, and was rapidly shown to be the main coreceptor for M-tropic human immunodeficiency virus (HIV)-1 strains and simian immunodeficiency virus (SIV). Chemokines constitute a rapidly growing family of proteins and receptor-chemokine interactions are known to be promiscuous and redundant. We have therefore tested whether other CC-chemokines could bind to and activate CCR5. All CC-chemokines currently available were tested for their ability to compete with [(125)I]-MIP-1beta binding on a stable cell line expressing recombinant CCR5, and/or to induce a functional response in these cells. We found that in addition to MIP-1beta, MIP-1alpha, and RANTES, five other CC-chemokines could compete for [(125)I]-MIP-1beta binding: MCP-2, MCP-3, MCP-4, MCP-1, and eotaxin binding was characterized by IC(50) values of 0.22, 2.14, 5.89, 29.9, and 21.7 nmol/L, respectively. Among these ligands, MCP-3 had the remarkable property of binding CCR5 with high affinity without eliciting a functional response, MCP-3 could also inhibit the activation of CCR5 by MIP-1beta and may therefore be considered as a natural antagonist for CCR5. It was unable to induce significant endocytosis of the receptor. Chemokines that could compete with high affinity for MIP-1beta binding could also compete for monomeric gp120 binding, although with variable potencies; maximal gp120 binding inhibition was 80% for MCP-2, but only 30% for MIP-1beta. MCP-3 could compete efficiently for gp120 binding but was, however, found to be a weak inhibitor of HIV infection, probably as a consequence of its inability to downregulate the receptor.
Assuntos
Quimiocinas CC/metabolismo , Citocinas , Proteínas Inflamatórias de Macrófagos/metabolismo , Proteínas Quimioatraentes de Monócitos/farmacologia , Receptores CCR5/metabolismo , Animais , Ligação Competitiva , Antagonistas dos Receptores CCR5 , Células CHO , Linhagem Celular , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL7 , Quimiocinas CC/farmacologia , Cricetinae , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Cinética , Receptores CCR5/imunologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , TransfecçãoRESUMO
Certain chemokine receptors serve as cofactors for HIV type 1 envelope (env)-mediated cell-cell fusion and virus infection of CD4-positive cells. Macrophage tropic (M-tropic) HIV-1 isolates use CCR5, and T cell tropic (T-tropic) strains use CXCR4. To investigate the cofactors used by simian immunodeficiency viruses (SIV), we tested four T-tropic and two M-tropic SIV env proteins for their ability to mediate cell-cell fusion with cells expressing CD4 and either human or nonhuman primate chemokine receptors. Unlike HIV-1, both M- and T-tropic SIV envs used CCR5 but not CXCR4 or the other chemokine receptors tested. However, by testing a panel of CCR5/CCR2b chimeras, we found that the structural requirements for CCR5 utilization by M-tropic and T-tropic SIV strains were different. T-tropic SIV strains required the second extracellular loop of CCR5 whereas a closely related M-tropic SIV strain could, like M-tropic HIV-1 strains, use the amino-terminal domain of CCR5. As few as two amino acid changes in the SIV env V3 domain affected the regions of CCR5 that were critical for fusogenic activity. Receptor signaling was not required for either fusion or infection. Our results suggest that viral tropism may be influenced not only by the coreceptors used by a given virus strain but also by how a given coreceptor is used.
Assuntos
Macrófagos/virologia , Receptores de Citocinas/imunologia , Receptores de HIV/imunologia , Transdução de Sinais , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia , Linfócitos T/virologia , Animais , Linhagem Celular , Humanos , Macrófagos/imunologia , Receptores CCR5 , Linfócitos T/imunologiaRESUMO
The chemokine receptor CCR5 is the major fusion coreceptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). To define the structures of CCR5 that can support envelope (Env)-mediated membrane fusion, we analyzed the activity of homologs, chimeras, and mutants of human CCR5 in a sensitive gene reporter cell-cell fusion assay. Simian, but not murine, homologs of CCR5 were fully active as HIV-1 fusion coreceptors. Chimeras between CCR5 and divergent chemokine receptors demonstrated the existence of two distinct regions of CCR5 that could be utilized for Env-mediated fusion, the amino-terminal domain and the extracellular loops. Dual-tropic Env proteins were particularly sensitive to alterations in the CCR5 amino-terminal domain, suggesting that this domain may play a pivotal role in the evolution of coreceptor usage in vivo. We identified individual residues in both functional regions, Asp-11, Lys-197, and Asp-276, that contribute to coreceptor function. Deletion of a highly conserved cytoplasmic motif rendered CCR5 incapable of signaling but did not abrogate its ability to function as a coreceptor, implying the independence of fusion and G-protein-mediated chemokine receptor signaling. Finally, we developed a novel monoclonal antibody to CCR5 to assist in future studies of CCR5 expression.
Assuntos
HIV-1/metabolismo , Receptores de Citocinas/metabolismo , Receptores de HIV/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Sítios de Ligação , DNA , Produtos do Gene env/metabolismo , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Primatas , Receptores CCR5 , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Macrophage-tropic (M-tropic) HIV-1 strains use the beta-chemokine receptor CCR5, but not CCR2b, as a cofactor for membrane fusion and infection, while the dual-tropic strain 89.6 uses both. CCR5/2b chimeras and mutants were used to map regions of CCR5 important for cofactor function and specificity. M-tropic strains required either the amino-terminal domain or the first extracellular loop of CCR5. A CCR2b chimera containing the first 20 N-terminal residues of CCR5 supported M-tropic envelope protein fusion. Amino-terminal truncations of CCR5/CCR2b chimeras indicated that residues 2-5 are important for M-tropic viruses, while 89.6 is dependent on residues 6-9. The identification of multiple functionally important regions in CCR5, coupled with differences in how CCR5 is used by M- and dual-tropic viruses, suggests that interactions between HIV-1 and entry cofactors are conformationally complex.
Assuntos
HIV-1/fisiologia , Conformação Proteica , Receptores de Quimiocinas , Receptores de Citocinas/química , Receptores de HIV/química , Sequência de Aminoácidos , Antígenos CD4/fisiologia , Efeito Citopatogênico Viral , Glicosilação , Células HeLa , Humanos , Substâncias Macromoleculares , Macrófagos/virologia , Fusão de Membrana , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Receptores CCR2 , Receptores CCR5 , Receptores de Citocinas/fisiologia , Receptores de HIV/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
Human immunodeficiency virus type 1 (HIV-1) requires both CD4 and a coreceptor to infect cells. Macrophage-tropic (M-tropic) HIV-1 strains utilize the chemokine receptor CCR5 in conjunction with CD4 to infect cells, while T-cell-tropic (T-tropic) strains generally utilize CXCR4 as a coreceptor. Some viruses can use both CCR5 and CXCR4 for virus entry (i.e., are dual-tropic), while other chemokine receptors can be used by a subset of virus strains. Due to the genetic diversity of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and the potential for chemokine receptors other than CCR5 or CXCR4 to influence viral pathogenesis, we tested a panel of 28 HIV-1, HIV-2, and SIV envelope (Env) proteins for the ability to utilize chemokine receptors, orphan receptors, and herpesvirus-encoded chemokine receptor homologs by membrane fusion and virus infection assays. While all Env proteins used either CCR5 or CXCR4 or both, several also used CCR3. Use of CCR3 was strongly dependent on its surface expression levels, with a larger number of viral Env proteins being able to utilize this coreceptor at the higher levels of surface expression. ChemR1, an orphan receptor recently shown to bind the CC chemokine I309 (and therefore renamed CCR8), was expressed in monocyte and lymphocyte cell populations and functioned as a coreceptor for diverse HIV-1, HIV-2, and SIV Env proteins. Use of ChemR1/CCR8 by SIV strains was dependent in part on V3 loop sequences. The orphan receptor V28 supported Env-mediated cell-cell fusion by four T- or dual-tropic HIV-1 and HIV-2 strains. Three additional orphan receptors failed to function for any of the 28 Env proteins tested. Likewise, five of six seven-transmembrane-domain receptors encoded by herpesviruses did not support Env-mediated membrane fusion. However, the chemokine receptor US28, encoded by cytomegalovirus, did support inefficient infection by two HIV-1 strains. These findings indicate that additional chemokine receptors can function as HIV and SIV coreceptors and that surface expression levels can strongly influence coreceptor use.
Assuntos
Quimiocinas CC , HIV-1/metabolismo , HIV-2/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores Virais/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Animais , Receptor 1 de Quimiocina CX3C , Quimiocina CCL1 , Células HeLa , Herpesviridae/metabolismo , Humanos , Fusão de Membrana , Receptores CCR3 , Receptores CCR8 , Receptores de Citocinas/metabolismo , Receptores de HIV/metabolismo , Células Tumorais CultivadasRESUMO
Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood-brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell-cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood-brain barrier disruption and viral entry into the central nervous system.