RESUMEN
The bicyclam AMD3100 (formula weight 830) blocks HIV-1 entry and membrane fusion via the CXCR4 co-receptor, but not via CCR5. AMD3100 prevents monoclonal antibody 12G5 from binding to CXCR4, but has no effect on binding of monoclonal antibody 2D7 to CCR5. It also inhibits binding of the CXC-chemokine, SDF-1alpha, to CXCR4 and subsequent signal transduction, but does not itself cause signaling and has no effect on RANTES signaling via CCR5. Thus, AMD3100 prevents CXCR4 functioning as both a HIV-1 co-receptor and a CXC-chemokine receptor. Development of small molecule inhibitors of HIV-1 entry is feasible.
Asunto(s)
Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD4-Positivos/virología , Quimiocinas CXC , VIH-1/fisiología , Compuestos Heterocíclicos/farmacología , Receptores CXCR4/fisiología , Anticuerpos Monoclonales/farmacología , Bencilaminas , Antígenos CD4/inmunología , Antígenos CD4/fisiología , Linfocitos T CD4-Positivos/efectos de los fármacos , Calcio/metabolismo , Carbacol/farmacología , Fusión Celular , Línea Celular , Células Cultivadas , Quimiocina CCL5/farmacología , Quimiocina CXCL12 , Ciclamas , Citocinas/metabolismo , Citocinas/farmacología , Proteína gp120 de Envoltorio del VIH/efectos de los fármacos , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/efectos de los fármacos , Humanos , Interleucina-2/farmacología , Cinética , Fusión de Membrana/efectos de los fármacos , Receptores CCR5/fisiología , Receptores CXCR4/efectos de los fármacos , Receptores CXCR4/inmunología , Transducción de Señal/efectos de los fármacos , Somatostatina/farmacologíaRESUMEN
The three-dimensional structure of the binding domain of the CD4 molecule has been determined and extensive mutational analyses of the respective binding sites on gp120 and CD4 have been completed. The consequences of gp120-CD4 binding with respect to secondary changes in the virion, or the cell, that may be required for infection or that may interfere with cellular function are current active areas of investigation.
Asunto(s)
Reacciones Antígeno-Anticuerpo , Antígenos CD4/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Secuencia de Aminoácidos , Antígenos CD4/genética , Linfocitos T CD4-Positivos/inmunología , Epítopos/genética , Proteína gp120 de Envoltorio del VIH/genética , Humanos , Datos de Secuencia Molecular , Estructura MolecularRESUMEN
CD4-based molecules were tested in combination with HIV-1-neutralizing antibodies directed against the V3 loop of gp120 or against gp41, for inhibition of HIV-1 envelope-mediated cell fusion. A virus-free cell fusion assay was developed, using Chinese hamster ovary cells that stably express HIV-1 gp120/gp41. These cells were incubated with dilutions of CD4-based molecules, antibodies, or mixtures of both, then overlaid with C8166 CD4+ T cells. Syncytia were counted and the degree of inhibition of cell fusion was determined. Synergy, additivity, or antagonism was calculated by the combination index (CI) method. The CD4-based molecules included soluble human CD4 as well as fusion proteins composed of CD4 linked to human immunoglobulin gamma 1 or gamma 2 heavy chains. Combinations of CD4-based molecules and monoclonal or polyclonal anti-V3 loop antibodies were synergistic in blocking HIV-1 envelope-mediated cell fusion (CI = 0.21-0.91 at 95% inhibition). Synergy was also observed with combinations of the CD4-based molecules and a broadly neutralizing anti-gp41 monoclonal antibody (2F5) (CI = 0.29-0.65 at 95% inhibition). These results demonstrate that molecules inhibiting HIV attachment act synergistically with molecules inhibiting HIV-1 fusion. The results suggest that CD4-based therapeutics would be more effective in patients with naturally occurring anti-V3 loop or anti-gp41 antibodies. In addition, there may be an advantage in coadministering CD4-based molecules and antibodies that block fusion, especially broadly neutralizing anti-gp41 antibodies, as a combination therapy for HIV-1 infections.
Asunto(s)
Antígenos CD4/farmacología , Inmunoadhesinas CD4/farmacología , Productos del Gen env/inmunología , Células Gigantes , Anticuerpos Anti-VIH/inmunología , VIH-1/patogenicidad , Secuencia de Aminoácidos , Animales , Células CHO , Fusión Celular , Cricetinae , Sinergismo Farmacológico , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Células HeLa , Humanos , Datos de Secuencia Molecular , Pruebas de Neutralización , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/farmacologíaRESUMEN
We tested human immunodeficiency virus type 1 (HIV-1) antibody-positive human sera and sCD4, alone and in combination, for synergistic, additive, or antagonistic effects on blocking of HIV binding and infectivity. Data were analyzed by an application of the median effect principle derived from the law of mass action. This allows the assessment of synergism/antagonism at any desired level of effect. Using three assays (whole virus binding to CD4 cells, neutralization of HIV infectivity, and binding of purified gp120 to solid-phase sCD4), we generally observed additive effects or slight synergism between antibody and sCD4 in inhibiting gp120-CD4 interaction. We used a fourth assay to measure the irreversible inactivation of HIV infectivity by sCD4, a property that can also be mediated by antibody but with considerably less potency than sCD4. The reduction in HIV infectivity mediated by mixtures of sCD4 and antibody was always equal to or greater than the arithmetic sum of the reductions by either agent alone. The relevant antiviral effects of sCD4 and anti-HIV sera may include reversible blockage of receptor binding, irreversible inactivation of HIV infectivity, and in the case of antibody, additional reactions that are independent of receptor binding. Although predictions concerning the in vivo situation are speculative, we find no evidence in vitro for antagonism between sCD4 and antibody with respect to the net effect of the two in blocking HIV binding and infectivity.
Asunto(s)
Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/microbiología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Sueros Inmunes/inmunología , Unión Competitiva , Sinergismo Farmacológico , VIH-1/inmunología , VIH-1/fisiología , Humanos , Masculino , Pruebas de NeutralizaciónRESUMEN
Primary isolates of human immunodeficiency virus type 1 (HIV-1) were obtained by coculture of peripheral blood lymphocytes (PBLs) from HIV-1-infected people with PBLs from uninfected donors. These viral stocks tend to be resistant to neutralization/inactivation by soluble CD4 (sCD4). When these stocks were passed through the T cell line C8166, virus stocks emerged that were sensitive to sCD4. Pre- and post-C8166 stocks maintained their sCD4-resistant and -sensitive phenotypes, respectively, with further passage in PBLs. Pre- and post-C8166 stocks were biologically cloned by two cycles of limiting dilution. The majority (14 of 17) of pre-C8166 clones were sCD4 resistant, and, conversely, the majority of post-C8166 clones (11 of 12) were sensitive to sCD4. Nucleotide and predicted amino acid sequence analysis in the env (gp120) region revealed a limited number of differences between the clones. The only differences that sorted with biological phenotype were in the first constant (C1) region of gp120. Adaptation to growth in C8166 cells and conversion from the sCD4-resistant to the sCD4-sensitive phenotype represent the emergence to prominence of viral species in the pre-C8166 stock that have a replication advantage in C8166 coincident with increased sensitivity to sCD4.
Asunto(s)
Antígenos CD/fisiología , Antígenos CD4/fisiología , Proteína gp120 de Envoltorio del VIH/genética , Seropositividad para VIH/virología , VIH-1/fisiología , Linfocitos/inmunología , Linfocitos/virología , Secuencia de Aminoácidos , Línea Celular , Células Cultivadas , ADN Viral/metabolismo , Células Gigantes , VIH-1/aislamiento & purificación , VIH-1/patogenicidad , Humanos , Cinética , Masculino , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Provirus/genética , Provirus/patogenicidad , Provirus/fisiologíaRESUMEN
Resistance-testing technology has been incorporated into the standard of care for human immunodeficiency virus type 1 (HIV-1) infection and therapy with protease and reverse transcriptase inhibitors. Inhibitors of HIV-1 entry represent an emerging mode of antiretroviral therapy, and HIV-1 entry inhibitors encompass three mechanistically distinct classes of agents known as attachment inhibitors, coreceptor inhibitors, and fusion inhibitors. Each class of agent has demonstrated promise in controlled clinical trials, and understanding the determinants and evolution of viral resistance will be critical for the optimal development and deployment of these new treatment classes. Advances in resistance-testing technologies have paralleled the development of HIV-1 entry inhibitor therapies, and the available data support the notion that attachment, coreceptor and fusion inhibitors offer complementary modes of therapy and distinct resistance profiles.
Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Amidas/farmacología , Bencilaminas , Inmunoadhesinas CD4/farmacología , Ciclamas , Farmacorresistencia Viral , Enfuvirtida , Proteína gp41 de Envoltorio del VIH/farmacología , Infecciones por VIH/virología , Compuestos Heterocíclicos/farmacología , Humanos , Fragmentos de Péptidos/farmacología , Piperazinas/farmacología , Compuestos de Amonio Cuaternario/farmacologíaAsunto(s)
Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Infecciones por VIH/microbiología , VIH/patogenicidad , Anticuerpos Monoclonales , Sitios de Unión , Antígenos CD4/genética , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Membrana Celular/metabolismo , Expresión Génica , Células HeLa/microbiología , Humanos , Activación de Linfocitos , Fusión de Membrana , Mutación , Ésteres del Forbol/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Tripsina/metabolismoRESUMEN
The binding of the human immunodeficiency virus envelope glycoprotein gp120 to the CD4 molecule is the initial step in the viral replicative cycle. This interaction is therefore an important target for therapeutic intervention for the treatment of human immunodeficiency virus infection. We designed an enzyme-linked immunosorbent assay which detects the interaction between recombinant soluble forms of CD4 and gp160. This assay could be used as an initial screen of libraries of synthetic chemical compounds and natural products.
Asunto(s)
Antígenos CD4/química , Proteína gp120 de Envoltorio del VIH/química , Línea Celular , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Infecciones por VIH/prevención & control , Humanos , Proteínas Recombinantes/química , Replicación Viral/efectos de los fármacosRESUMEN
CD4 (T4), a glycoprotein expressed largely on the surface of cells in the immune system, serves as the receptor for the human immunodeficiency virus, HIV. The isolation of the CD4 gene has permitted an analysis of the structure of CD4 and its role in both HIV infection and the immune response. Recently, new classes of CD4-based therapeutics have been generated that interfere with HIV attachment to target cells. Soluble CD4 proteins and CD4-based chimeric molecules are currently undergoing clinical evaluation in HIV-infected individuals.
Asunto(s)
Antígenos CD4/fisiología , Infecciones por VIH/inmunología , Sitios de Unión , Antígenos CD4/análisis , Antígenos CD4/genética , Linfocitos T CD4-Positivos/microbiología , VIH/fisiología , Humanos , Transducción de Señal , Linfocitos T/inmunologíaRESUMEN
The T cell surface glycoproteins T4 and T8 are thought to mediate efficient cell-cell interactions in the immune system and in this way may be responsible for the appropriate targeting of subpopulations of T cells. We have used gene transfer combined with subtractive hybridization to isolate both cDNA and functional genomic clones encoding the T8 protein. The sequence of the cDNA reveals that T8 is a transmembrane protein with an N-terminal domain which shares significant homology to immunoglobulin variable region light chains. This immunoglobulin-like structure is likely to be important in the function of T8 during differentiation and in the course of the immune response.
Asunto(s)
Genes , Glicoproteínas/genética , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos/análisis , Secuencia de Bases , ADN/aislamiento & purificación , Glicoproteínas/fisiología , Humanos , Células L/inmunología , Complejo Mayor de Histocompatibilidad , Ratones , Hibridación de Ácido Nucleico , Transformación GenéticaRESUMEN
Receptor binding of HIV to the CD4 molecule is required for efficient infection of T cells, but the post-binding steps that result in penetration of HIV are not well understood. CD4 is induced to internalize upon T cell activation, and mAb to CD4 modify signal transduction and T cell activation as does HIV in some systems. It is not known whether HIV binding triggers CD4 endocytosis or whether signal transduction events are required for penetration. Selected inhibitors of signal transduction were evaluated for their effects on penetration using two assays that are dependent on penetration. After short term exposure to inhibitor and HIV, cells were analyzed for reverse-transcribed HIV DNA (DNA amplification assay), or productive infection is monitored (infectivity assay). Viral penetration was tested in the presence of H7 (protein kinase C inhibition), EGTA (extracellular Ca2+ chelation), cyclosporine A (inhibition of Ca2+/calmodulin-dependent activation), or pertussis toxin (inhibition of G protein function). All agents were used at concentrations that were inhibitory for their respective signal transduction pathways. None of the inhibitors affected viral penetration. We tracked the CD4 molecule with fluorescent probes that do not interfere with HIV binding in a system where CD4 T cells were saturated with HIV and the penetration event was relatively synchronized. Under conditions where detection of CD4 was more sensitive than the detection of HIV, HIV internalization was readily detected but CD4 internalization was not.
Asunto(s)
Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , VIH-1/patogenicidad , Sulfonamidas , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Calcio/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Calmodulina/fisiología , Células Cultivadas , Ciclosporinas/farmacología , Citocalasinas/farmacología , Ácido Egtácico/farmacología , VIH-1/inmunología , Humanos , Técnicas In Vitro , Isoquinolinas/farmacología , Activación de Linfocitos , Proteínas del Tejido Nervioso/fisiología , Toxina del Pertussis , Piperazinas/farmacología , Reacción en Cadena de la Polimerasa , Proteína Quinasa C/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The isolation of clones encoding the human surface protein T4, and the expression of the T4 gene in new cellular environments, have enabled us to examine the role of this protein in the pathogenesis of AIDS. Our studies support a mechanism of AIDS virus infection that initially involves the specific interaction of the AIDS virus with T4 molecules on the cell surface. This association can be demonstrated on T4+ transformed T and B lymphocytes as well as epithelial cells. Furthermore, the presence of T4 on the surface of all human cells examined is sufficient to render these cells susceptible to AIDS virus infection. Our data suggest that the T4-AIDS virus complex is then internalized by receptor-mediated endocytosis. Finally, we find that the T4 gene is expressed in the brain as well as in lymphoid cells, providing an explanation for the dual neurotropic and lymphotropic character of the AIDS virus. In this manner, a T lymphocyte surface protein important in mediating effector cell-target cell interactions has been exploited by a human retrovirus to specifically target the AIDS virus to populations of T4+ cells.
Asunto(s)
Antígenos de Superficie/genética , Encéfalo/metabolismo , VIH/metabolismo , Sistema Inmunológico , Receptores Virales/metabolismo , Cloruro de Amonio/farmacología , Animales , Antígenos de Diferenciación de Linfocitos T , Linfocitos B/microbiología , Transformación Celular Viral , Efecto Citopatogénico Viral , Endocitosis , Citometría de Flujo , VIH/patogenicidad , Células HeLa , Humanos , Ratones , Mitomicina , Mitomicinas/farmacología , Linfocitos T/microbiologíaRESUMEN
The surface glycoproteins T4 and T8 define functionally distinct populations of T lymphocytes. We have obtained cDNA and genomic clones encoding the T4 molecule and used these as probes to determine the chromosomal location of this gene. Genomic blotting experiments, along with in situ hybridization analyses, indicate that the T4 gene resides on the short arm of human chromosome 12, at region p12-pter. Thus, the T4 gene is not linked to any known member of the immunoglobulin gene family, including its counterpart gene, T8, which resides on human chromosome 2 immediately distal to the immunoglobulin kappa locus.
Asunto(s)
Antígenos de Superficie/genética , Cromosomas Humanos 6-12 y X , Proteínas de la Membrana/genética , Receptores Virales/genética , Linfocitos T/fisiología , Antígenos de Diferenciación de Linfocitos T , Mapeo Cromosómico , ADN/genética , Humanos , Células Híbridas , Hibridación de Ácido NucleicoRESUMEN
Human immunodeficiency virus type 1 (HIV-1) entry proceeds via a cascade of events that afford promising targets for therapy. PRO 542 neutralizes HIV-1 by blocking its attachment to CD4 cells, and T-20 blocks gp41-mediated fusion. Both drugs have shown promise in phase 1/2 clinical trials. Here, the drugs were tested individually and in combination in preclinical models of HIV-1 infection, and inhibition data were analyzed for cooperativity by using the combination index method. Synergistic inhibition of virus-cell and cell-cell fusion was observed for phenotypically diverse viruses for a broad range of drug concentrations, often resulting in > or = 10-fold dose reductions in vitro. Additional mechanism-of-action studies probed the molecular basis of the synergies. The markedly enhanced activity observed for the PRO 542:T-20 combination indicates that the multistep nature of HIV-1 entry leaves the virus particularly vulnerable to combinations of entry inhibitors. These findings provide a strong rationale for evaluating combinations of these promising agents for therapy in vivo.
Asunto(s)
Fármacos Anti-VIH/farmacología , Inmunoadhesinas CD4/farmacología , Proteína gp41 de Envoltorio del VIH/farmacología , VIH-1/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Animales , Células CHO , Fusión Celular , Línea Celular , Cricetinae , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Enfuvirtida , Células Eucariotas/patología , Células Eucariotas/virología , Infecciones por VIH/virología , VIH-1/fisiología , Células HeLa , Humanos , Linfocitos T/virología , Replicación Viral/efectos de los fármacosRESUMEN
CD4-immunoglobulin G2 (IgG2) is a fusion protein comprising human IgG2 in which the Fv portions of both heavy and light chains have been replaced by the V1 and V2 domains of human CD4. Previous studies found that CD4-IgG2 potently neutralizes a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates in vitro and ex vivo. The current report demonstrates that CD4-IgG2 protects against infection by primary isolates of HIV-1 in vivo, using the hu-PBL-SCID mouse model. Passive administration of 10 mg of CD4-IgG2 per kg of body weight protected all animals against subsequent challenge with 10 mouse infectious doses of the laboratory-adapted T-cell-tropic isolate HIV-1(LAI), while 50 mg of CD4-IgG2 per kg protected four of five mice against the primary isolates HIV-1(JR-CSF) and HIV-1(AD6). In contrast, a polyclonal HIV-1 Ig fraction exhibited partial protection against HIV-1(LAI) at 150 mg/kg but no significant protection against the primary HIV-1 isolates. The results demonstrate that CD4-IgG2 effectively neutralizes primary HIV-1 isolates in vivo and can prevent the initiation of infection by these viruses.
Asunto(s)
Fármacos Anti-VIH/inmunología , Inmunoadhesinas CD4/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , Animales , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacocinética , Inmunoadhesinas CD4/administración & dosificación , Células CHO , Cricetinae , Modelos Animales de Enfermedad , Anticuerpos Anti-VIH/inmunología , VIH-1/aislamiento & purificación , Humanos , Inmunoglobulina G/inmunología , Ratones , Ratones SCIDRESUMEN
We tested the ability of human monoclonal antibodies (immunoglobulin G1b12 [IgG1b12] and 19b) and CD4-based molecules (CD4-IgG2 and soluble CD4 [sCD4]) to neutralize human immunodeficiency virus type 1 directly from the plasma of seropositive donors in an ex vivo neutralization assay. IgG1b12 and CD4-IgG2, at concentrations from 1 to 25 micrograms/ml, were found to be effective at reducing the HIV-1 titer in most plasma samples. When viruses recovered from plasma samples were expanded to produce virus stocks, no correlation between the neutralization sensitivities to IgG1b12 and CD4-IgG2 of the in vitro passaged stocks and those of the ex vivo neutralizations performed directly on the plasma was observed. These differences could be due to changes in neutralization sensitivity that occur after one passage of the virus in vitro, or they could be related to the presence of complement or antibodies in the plasma. Furthermore, differences in expression of adhesion molecules on plasma-derived and phytohemagglutinin-activated peripheral blood mononuclear cell-derived viruses could be involved. These studies suggest that IgG1b12 and CD4-IgG2 have broad and potent neutralizing activity in both in vitro and ex vivo neutralization assays and should be considered for use as potential immunoprophylactic or therapeutic agents.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Antivirales/farmacología , Antígenos CD4/farmacología , Inmunoadhesinas CD4/farmacología , Seropositividad para VIH/sangre , Seropositividad para VIH/inmunología , Seropositividad para VIH/virología , VIH-1/efectos de los fármacos , Humanos , Inmunoglobulina G/inmunología , Pruebas de Neutralización , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacologíaRESUMEN
The surface glycoproteins T4 and T8 define different functional subsets of T lymphocytes and may act as recognition molecules mediating appropriate interactions between the T cell and its target. Previously we employed gene transfer and subtractive hybridization to isolate a T8 cDNA; now we have isolated and sequenced a cDNA clone encoding the T4 molecule. The deduced protein sequence reveals that T4 is an integral membrane protein that shares significant amino acid and structural homologies with members of the immunoglobulin supergene family. The overall structure of T4 consists of an N-terminal variable (V)-like domain, a joining (J)-like region, a third extracellular domain, a membrane-spanning region homologous to class II MHC beta-chains, and a highly charged cytoplasmic domain. Comparison of the protein sequences deduced from the T4 and T8 cDNAs reveals structural similarities consistent with their postulated role as recognition molecules, as well as differences suggesting that the two proteins recognize different structures on the target cell.
Asunto(s)
Antígenos de Superficie/genética , ADN , Inmunoglobulinas/genética , Proteínas de la Membrana/genética , Linfocitos T/análisis , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación de Linfocitos T , Secuencia de Bases , Línea Celular , ADN/aislamiento & purificación , Deltaretrovirus , Genes , Glicoproteínas/genética , Glicoproteínas/fisiología , Humanos , Proteínas de la Membrana/fisiología , Ratones , Hibridación de Ácido Nucleico , Receptores Virales , Linfocitos T/inmunología , Transformación GenéticaRESUMEN
Two assays for measuring inhibition of human immunodeficiency virus type 1 (HIV-1) infection by soluble CD4 (sCD4) are described. Experiments in which sCD4, HIV-1, and cell concentrations and sequence of combination, noninfectious/infectious particle ratio, and temperature were varied produced results that support the conclusion that sCD4 inhibits HIV-1 infection by two mechanisms: reversible blockage of receptor binding and irreversible inactivation of infectivity. Fresh isolates obtained from HIV-1-infected persons were tested in both assays and found to be more resistant to both mechanisms of sCD4-mediated inhibition than multiply passaged laboratory strains. Binding studies revealed similar affinities for sCD4 in detergent lysates of sensitive and resistant strains at both 4 and 37 degrees C. The avidity of intact virions for sCD4 was lower at 4 than at 37 degrees C, and in the presence of excess sCD4, less sCD4 was bound at 4 than at 37 degrees C. The avidity differences were similar for fresh isolates and laboratory strains. However, fresh isolates were more resistant to sCD4-induced shedding of envelope glycoprotein gp120 from intact virions than was the laboratory strain. Relative resistance to sCD4 by certain isolates does not represent a lower intrinsic affinity of their envelope for sCD4 or a lower capacity for sCD4 binding. Rather, an event that occurs after binding may account for the differences. This postbinding event or feature may be determined by regions of the envelope outside the CD4 binding site.
Asunto(s)
Antígenos CD4/farmacología , Infecciones por VIH/inmunología , VIH-1/efectos de los fármacos , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos , Línea Celular , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/crecimiento & desarrollo , VIH-1/inmunología , VIH-1/aislamiento & purificación , Humanos , Linfocitos/microbiología , Pruebas de Neutralización , Pruebas de Precipitina , Receptores del VIH/metabolismo , Sensibilidad y Especificidad , Pase Seriado , Proteínas Virales/análisis , Proteínas Virales/metabolismo , Virión/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
The T4 molecule (CD4) is an important component of the human immunodeficiency virus (HIV) receptor. As yet, no other component has been demonstrated. We report here that two cell lines, a B lymphoblastoid cell line (Gupta) and a glial cell line (HEB) derived from human embryonal brain tissue, are productively infectable with two distinct isolates of HIV as judged by electron microscopy and immunological and virological studies. These two cell lines do not display detectable surface CD4 glycoprotein. However, using S1 nuclease analysis, we have found that both cell lines do express low levels of CD4 mRNA. Neither of them produced syncytia formation upon HIV infection, a recognized feature of HIV-infected cells strongly expressing the CD4 glycoprotein. It is conceivable that the CD4 mRNA is translated, resulting in meager surface expression of CD4 molecules undetectable by conventional techniques. Therefore, infection with HIV may be one of the most sensitive methods of demonstrating low levels of CD4 expression by human cells. Furthermore, HIV-infected Gupta cells have here been shown to be more susceptible to the lytic activity of natural killer (NK) cells than their uninfected counterparts. These phenomena may be important for pathogenesis of HIV-associated disorders.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Linfocitos B/microbiología , VIH/crecimiento & desarrollo , Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos de Diferenciación de Linfocitos T/genética , Northern Blotting , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD4-Positivos/fisiología , Línea Celular , Citotoxicidad Inmunológica , Endonucleasas/metabolismo , Citometría de Flujo , VIH/genética , Humanos , Inmunidad Celular , Células Asesinas Naturales/inmunología , ARN Mensajero/genética , Receptores Virales/fisiología , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Replicación ViralRESUMEN
CD4 (T4) is a glycoprotein of relative molecular mass 55,000 (Mr 55K) on the surface of T lymphocytes which is thought to interact with class II MHC (major histocompatibility complex) molecules, mediating efficient association of helper T cells with antigen-bearing targets. The CD4 protein is also the receptor for HIV, a T-lymphotropic RNA virus responsible for the human acquired immune deficiency syndrome (AIDS) (refs 4-7). To define the mechanisms of interaction of CD4 with the surface of antigen-presenting cells and with HIV, we have isolated the CD4 gene and expressed this gene in several different cellular environments. Here we describe an efficient expression system in which a recombinant, soluble form of CD4 (sCD4) is secreted into tissue culture supernatants. This sCD4 retains the structural and biological properties of CD4 on the cell surface, binds to the envelope glycoprotein (gp110) of HIV and inhibits the binding of virus to CD4+ lymphocytes, resulting in a striking inhibition of virus infectivity.