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W C Greene

Publications and source records attributed to W C Greene.

At least 127 records · Page 7Linked to original sources
PubMed1988

Functional replacement of the HIV-1 rev protein by the HTLV-1 rex protein.

Two evolutionarily distinct families of human retroviruses, the human immunodeficiency viruses (HIV) and the human T-cell leukaemia viruses (HTLV), have been defined (reviewed in ref. 1). Although these virus groups share tropism for human CD4+ T cells, they differ markedly in primary sequence, genetic organization and disease association (AIDS versus adult T-cell leukaemia), but show similar general strategies for the regulation of viral gene expression. Each encodes a protein able to trans-activate transcription from the homologous viral long terminal repeat (tat in HIV, tax in HTLV), although these proteins act by different mechanisms and do not appear to be interchangeable. Each virus also produces a second trans-acting protein that induces the expression of the unspliced messenger RNAs encoding the viral structural proteins (rev in HIV and rex in HTLV). Here we show that the rex protein of HTLV-I can functionally replace the rev protein of HIV-1 in transient expression assays. This genetic complementation by rex is adequate for the rescue of a replication-defective rev mutant of HIV-1. This unexpected shared function between the structurally distinct rex and rev proteins emphasizes the importance of this highly conserved pathway for the regulation of human retrovirus gene expression.

Gene Expression Regulation↗
PubMed1988

HTLV-I tax induces cellular proteins that activate the kappa B element in the IL-2 receptor alpha gene.

Jurkat T cell lines constitutively expressing Tax, the 40-kilodalton transactivator protein of human T lymphotropic virus type I (HTLV-I), were used to investigate the mechanism by which this viral product deregulates the expression of the interleukin-2 receptor alpha gene (IL-2R alpha, Tac). Transfection of deleted forms of the IL-2R alpha promoter and in vitro DNA-binding studies revealed that a 12-base pair promoter segment, which has homology with the binding site for NF-kappa B, was required for Tax-induced activation of the IL-2R alpha promoter in vivo. An 18-base pair oligonucleotide containing this kappa B-like regulatory element proved sufficient to confer Tax inducibility upon a heterologous promoter. DNA affinity precipitation assays showed that Tax, like mitogenic stimuli, induced the expression of the 86-kilodalton cellular protein HIVEN86A, which specifically binds to the IL-2R alpha kappa B element in vitro. Furthermore, DNA/protein cross-linking studies revealed that several polypeptides interact with this sequence motif. Thus, the deregulation of IL-2R alpha gene expression encountered in HTLV-I leukemias appears to involve Tax activation of one or more cellular proteins that are normally induced by mitogens and that directly contribute to transcriptional activation of this receptor gene.

Acetyltransferases↗
PubMed1988

The same inducible nuclear proteins regulates mitogen activation of both the interleukin-2 receptor-alpha gene and type 1 HIV.

Both the interleukin-2 receptor-alpha (Tac, p55, IL-2R alpha) gene and the long terminal repeat (LTR) of type 1 HIV are activated by various T cell mitogens. We now demonstrate that an inducible 86 kd nuclear protein termed HIVEN86A specifically binds to both the enhancer element of the HIV-1 LTR and a closely related 12 bp sequence present in the 5' regulatory region (-267 to -256) of the IL-2R alpha gene. The interaction of these binding sites with HIVEN86A and perhaps other cellular proteins such as NF-kappa B appears importantly involved in mitogen activation since the isolated IL-2R alpha promoter binding site or single elements of the normally duplicated HIV-1 enhancer alone are sufficient to confer mitogen inducibility on an unresponsive heterologous promoter. Together these findings suggest that the normal action of an inducible nuclear DNA binding protein(s) involved in the regulation of IL-2R alpha gene expression can be subverted by the HIV-1 provirus to promote activation of retroviral gene transcription.

Base Sequence↗
PubMed1988

Sequence requirements for ligand binding and cell surface expression of the Tac antigen, a human interleukin-2 receptor.

Discrete peptide domains within the primary sequence of cell-surface receptor glycoproteins are believed to regulate not only their function but also their targeting to the cell membrane. To identify sequence elements required for intracellular transport and ligand binding by the human Tac interleukin-2 (IL-2) receptor, we prepared expression plasmids encoding a series of artificially mutated or naturally occurring variants of the Tac cDNA. In particular, we sought to further delineate the functional role of the sequences contributed by each of the eight exons that together encode the Tac protein. Deletion of exons 5 through 8 of the receptor had no detectable effect on IL-2 binding or intracellular transport of the Tac protein, and resulted in secreted forms of this IL-2-binding protein. Removal of sequences corresponding to all of exon 4 ablated IL-2 binding activity yet still permitted transport to the cell surface. In contrast, partial deletion of exon 4 sequences resulted in proteins that not only lacked IL-2 binding activity but also were sequestered within the endoplasmic reticulum. Removal of one or both of the N-linked glycosylation sites present in the Tac protein did not impair receptor transport or ligand binding. These results demonstrate that exon 4 of the Tac gene encodes amino acid residues that play an important role in regulating both the intracellular transport and function of this IL-2 receptor.

Animals↗
PubMed1988

Lymphocytes isolated from the intestinal lamina propria of normal nonhuman primates have increased expression of genes associated with T-cell activation.

Synthesis of interleukin-2 (IL-2) and expression of interleukin-2 receptors play central roles in T-cell activation, proliferation, and differentiation. The state of activation of T lymphocytes in the intestinal lamina propria was compared with that of circulating lymphocytes and lymphocytes isolated from the spleen or mesenteric lymph nodes of normal nonhuman primates. Lamina propria lymphocytes (LPL) had significantly higher proliferation in response to recombinant IL-2 compared with the other populations. In agreement with this finding, LPL had a significantly higher percentage of interleukin-2 receptor-positive (IL-2R+) cells as determined by staining with fluoresceinated monoclonal anti-IL-2R antibody. Two-color immunofluorescence staining showed that both CD4+ and CD8+ lamina propria T cells were IL-2R+. It was also found that the percentage of major histocompatibility complex class II-positive T cells was higher in the lamina propria. Northern blot analysis with a cDNA specific for the IL-2R showed that unstimulated LPL had easily detectable IL-2R mRNA, whereas no IL-2R mRNA was found in unstimulated lymphocyte populations from other sites. The activation of the IL-2R gene in LPL was not associated with the activation of other cellular genes (actin, major histocompatibility complex class I). Although no IL-2 bioactivity was measured in culture supernatants of unstimulated lymphocytes, concanavalin A-stimulated LPL produced significantly more IL-2 than other lymphocytes. This finding was confirmed at the molecular level as IL-2 mRNA was not detected in unstimulated LPL but was found in concanavalin A-stimulated LPL. Thus, normal lamina propria T lymphocytes have selective expression of genes associated with cell activation.

Animals↗
PubMed1988

Regulation of interleukin 2 receptor alpha subunit (Tac or CD25 antigen) gene expression: binding of inducible nuclear proteins to discrete promoter sequences correlates with transcriptional activation.

Transfection of deleted forms of the human interleukin 2 receptor alpha subunit (IL-2R alpha; also called CD25 or Tac antigen) gene (IL2RA) promoter revealed a requirement for sequences 3' of base -317 for phytohemagglutinin- and phorbol 12-myristate 13-acetate (PMA)-induced promoter activation in CD4+ Jurkat T cells. In contrast, sequences 3' of base -271 were sufficient for promoter induction in CD4-/CD8- YT-1 T cells or Jurkat cells expressing the transactivator protein (tat-I) of human T-cell lymphotropic virus type I (HTLV-I). Gel retardation assays revealed that nuclear extracts from induced, but not uninduced, Jurkat and YT-1 cells mediated the formation of two specific DNA-protein complexes with oligonucleotides spanning the region of the IL2RA promoter from position -291 to -245, which contains two imperfect direct repeats (IDRs). Consistent with the different 5' sequence requirements for promoter activation in Jurkat and YT-1 cells, oligonucleotides corresponding to the region from -267 to -243 (downstream IDR and flanking region) formed only one complex with induced Jurkat extracts but two complexes with induced YT-1 extracts. Oligonucleotides containing the region of the IL2RA promoter from -293 to -270 (upstream IDR and flanking region) failed to bind protein in either cell type. In further support of the biological significance of these DNA-protein interactions, the IL2RA oligonucleotide from -291 to -245 proved to be sufficient in either orientation to confer PMA inducibility to the mitogen-insensitive thymidine kinase gene promoter in Jurkat cells. Together, these findings suggest that the interaction of inducible DNA binding proteins with the IL2RA promoter between bases -291 and -245 plays an important role in mitogen-induced changes in the transcriptional activity of this receptor gene. Furthermore, the requisite 5' sequences appear to differ in T cells depending upon the nature of the activation signal and perhaps the stage of cellular maturation.

Base Sequence↗
PubMed1988

Identification of specific residues of human interleukin 2 that affect binding to the 70-kDa subunit (p70) of the interleukin 2 receptor.

Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70). Substitution of Asp20 or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high-affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit. The presence of the carboxylic acid in the side chain of Asp20 was necessary for effective binding to the p70 protein. In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays. The effects of these mutations on protein conformation were assessed by circular dichroism. These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity.

Binding, Competitive↗
PubMed1988

Stable expression of the tax gene of type I human T-cell leukemia virus in human T cells activates specific cellular genes involved in growth.

Stable expression of the 40-kDa transactivator protein (Tax) from the type I human T-cell leukemia virus (HTLV-I) in Jurkat T cells leads to the activation and sustained expression of certain cellular genes that are transiently induced during normal T-cell growth. Cellular genes induced by Tax include those encoding the alpha subunit of the high-affinity interleukin 2 receptor (Tac), interleukin 2, and granulocyte/macrophage colony-stimulating factor. Tax induction of the interleukin 2 gene is synergistically amplified by mitogens that augment cytoplasmic levels of calcium. These changes in the pattern of cellular gene expression reflect a specific action of Tax, as they are undetectable in isogenically matched control cell lines expressing antisense tax cDNA. The spectrum of cellular genes regulated by Tax appears to be restricted: several other T-cell genes, either inducibly or constitutively expressed, are unaffected by this viral protein. These cell lines constitutively expressing Tax provide valuable reagents to explore the molecular basis for Tax action and to delineate the full spectrum of cellular genes regulated by this retroviral gene product.

Animals↗
PubMed1988

Novel interleukin 2 (IL-2) receptor appears to mediate IL-2-induced activation of natural killer cells.

A novel IL-2 receptor, distinct from the Tac protein, has been identified on the surface of purified human natural killer (NK) cells by chemical cross-linking of 125I-IL-2. This protein is approximately 70,000 D in size (p70) and appears to be identical to the recently recognized second subunit of the human high affinity IL-2 receptor complex. Scatchard analysis of 125I-IL-2 binding to purified NK cells revealed approximately 2,300 p70 binding sites per cell with an apparent dissociation constant of 200 pM, a value intermediate between the previously recognized high and low affinity forms of the human IL-2 receptor. The monoclonal anti-Tac antibody did not inhibit the cross-linking of 125I-IL-2 to the p70 binding sites present on NK cells. Functionally, the addition of high concentrations of recombinant IL-2 to the enriched NK cells promoted a rapid augmentation of cytolytic activity and a more delayed increase in cellular proliferation. Anti-Tac effectively blocked the IL-2-induced proliferative response in these cells, but failed to alter the enhancement of cytotoxicity. Analysis of NK cytoplasmic RNA isolated at various time points after IL-2 stimulation revealed the rapid induction of c-myb and Tac gene expression that was also not inhibited by the anti-Tac antibody. These findings suggest that IL-2 binding to the p70 receptor constitutively expressed on the surface of NK cells may mediate both the development of increased cytolytic activity and rapid changes in gene expression. The activation of the Tac gene may in turn permit the formation of the high affinity IL-2 receptor complex (comprised of at least the Tac and p70 proteins) that appears to transduce the requisite signals involved in NK cell proliferation.

Antigens, Surface↗
PubMed1987

Activation of the HIV-1 LTR by T cell mitogens and the trans-activator protein of HTLV-I.

To investigate the mechanism by which immune activation augments replication of the human immunodeficiency virus type 1 (HIV-1) in infected T cells, four different classes of T cell mitogens were evaluated for their effects on the HIV-1 long terminal repeat (LTR). Phytohemagglutinin (PHA), a mitogenic lectin; phorbol 12-myristic 13-acetate, a tumor promoter; ionomycin, a calcium ionophore; and tat-1, the trans-activator protein from the human T cell leukemia/lymphoma virus type I (HTLV-I) each stimulated the HIV-1 LTR. Studies of deleted forms of the LTR supported a central role in these responses for the HIV-1 enhancer, which alone was sufficient for mitogen inducibility, but also suggested that other 5' positive and negative regulatory elements contribute to the overall magnitude of the response. Synergistic activation of the HIV-1 LTR (up to several thousandfold) was observed with combinations of these mitogens and the HIV-1--derived tat-III protein. Cyclosporin A, an immunosuppressive agent, inhibited PHA-mediated activation of the HIV-1 LTR but was without effect in the presence of other mitogens. Thus, HIV-1 gene expression and replication appear to be regulated, via the HIV-1 LTR, by the same mitogenic signals that induce T cell activation.

Cell Line↗
PubMed1987

Contrasting interleukin 2 binding properties of the alpha (p55) and beta (p70) protein subunits of the human high-affinity interleukin 2 receptor.

In this report, we have investigated the kinetics of IL-2 binding to the alpha (p55) and beta (p70) IL-2 binding proteins and compared these properties with ligand binding to the high-affinity IL-2-R. The association and dissociation of IL-2 to the alpha (p55) chain occurred with very rapid kinetics (t 1/2 = 4-10 s). In contrast, IL-2 association to, and dissociation from the beta (p70) chain occurred at a greatly reduced rate (t 1/2 = 40-50 min and 200-400 min, respectively). Measurements of IL-2 binding to the high-affinity receptor revealed an interesting composite of these binding properties with a rapid association rate (t 1/2 = 30-45 s) resembling the alpha (p55) chain and a slow dissociation rate (t 1/2 = 270-300 min) similar to the beta (p70) chain. These findings provide additional support for the model of the high-affinity IL-2-R as a heterodimeric membrane complex composed of both the alpha (p55) and beta (p70) subunits and suggest that high-affinity IL-2 binding may involve a conformational change in structure of either or possibly both of the receptor chains. These results highlight the important and perhaps different role played by each subunit in the formation of functional high-affinity IL-2-R.

Binding Sites↗
PubMed1987

Reconstitution of a functional interleukin 2 receptor in a nonlymphoid cell.

The maintenance of T lymphocytes which are important effectors of immune responses requires the T cell growth factor, interleukin 2 (IL-2). The binding of IL-2 to specific cell-surface receptors (IL-2R) has previously been shown to be essential to the growth and proliferation of activated lymphocytes. A human IL-2R cDNA sequence, placed under the control of the SV40 transcriptional promoter and enhancer, has been transfected into murine L cells. Single cell analysis by autoradiography was used to show that fibroblastic L cells, stably expressing human IL-2R, respond to stimulation with IL-2 by DNA synthesis and proliferation. This response is specifically blocked by the addition of an anti-IL-2R monoclonal antibody, anti-Tac, as previously reported. Neither nonspecific antisera nor 7G7/B6, an anti-IL-2R monoclonal antibody which does not interfere with IL-2 binding to its receptor, had any effect on this response. The induction of DNA synthesis by IL-2 is both rapid and dose-dependent. The ability of IL-2 to stimulate these transfected L cells to proliferate demonstrates that a lymphoid environment is not required for the functional interaction between IL-2 and its receptor, and provides a unique model system for the investigation of the molecular basis for the cellular events mediated by IL-2.

Animals↗
PubMed1987

Direct activation of human resting T cells by IL 2: the role of an IL 2 receptor distinct from the Tac protein.

High concentrations of interleukin 2 (IL 2) were shown to produce a delayed but pronounced proliferation of purified resting T cells in the apparent absence of other activation signals. Because these stimulatory effects of IL 2 occurred in the absence of detectable Tac+ cells, the possibility that IL 2 might be initially interacting with an IL 2 binding protein distinct from the Tac protein was studied. Chemical cross-linking studies with 125I-IL 2 revealed the presence of an IL 2 binding protein distinct from the Tac protein on the surface of these unstimulated T cells. This second IL 2 receptor has an estimated molecular size of 70,000 daltons, lacks reactivity with the anti-Tac antibody, and appears to be identical to the p70 protein recently proposed as a component of the high affinity IL 2 receptor. Scatchard analysis of IL 2 binding assays performed with the unactivated T cells revealed approximately 600 to 700 p70 sites per cell and an apparent Kd of 340 pM. These data indicate that the p70 protein present on resting T cells binds IL 2 with an intermediate affinity compared with the previously recognized high and low affinity forms of the receptor and may account for the high concentration of IL 2 needed to induce resting T cell proliferation. To investigate the early biologic consequences of IL 2 binding to the p70 protein, potential changes in the expression of genes involved in T cell activation were examined. Northern blotting revealed the rapid induction of c-myc, c-myb, and Tac mRNA after stimulation of resting T cells with a high concentration of IL 2. The anti-Tac antibody did not inhibit IL 2 induced expression of these genes, suggesting that the p70 protein rather than the Tac antigen or the high affinity IL 2 receptor complex mediated this signal. However, in contrast to these early activation events, the anti-Tac antibody significantly inhibited IL 2 induced T cell proliferation. This finding implicates the high affinity form of the IL 2 receptor in the proliferative response of the IL 2 activated T cells. Thus these data support a two step model for the induction of resting T cell proliferation by high doses of IL 2 involving the initial generation of an activation or "competence" signal through the p70 protein and a subsequent proliferation or "progression" signal through the high affinity form of the receptor.

Antigens, Surface↗
PubMed1987

Expression of functional IL 2 receptors by lipopolysaccharide and interferon-gamma stimulated human monocytes.

Human peripheral blood monocytes were stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) alone or in combination. Stimulated but not resting monocytes displayed the Tac peptide of the interleukin 2 (IL 2) receptor within 24 hr as measured by immunofluorescence staining and [3H] Tac binding. The total number of anti-Tac binding sites on co-stimulated monocytes was 13,700. By using scatchard analysis with radiolabeled IL 2, the activated cells were shown to express low numbers (below 100 sites/cell) of high affinity binding sites with a KD of approximately 15 pM. LPS and IFN-gamma were additive in augmenting the number of IL 2 and anti-Tac binding sites. By using an ELISA assay specific for the soluble released form of the Tac peptide we identified 112 U/ml of IL 2 receptors in the supernatant of monocytes stimulated for 24 hr with IFN-gamma, 233 U/ml after stimulation with LPS, and 519 U/ml after the addition of both stimulating agents. Both the membrane form (55,000 daltons), as well as the soluble form (45,000 to 50,000 daltons) of the Tac, IL 2 receptor, peptide from monocytes were shown by immunoprecipitation and gel electrophoresis to be similar size to the comparable forms of these receptors derived from activated T cells. In addition, monocytes stimulated for 8 hr contained mRNA specifically hybridizing to a cDNA probe coding for the Tac peptide. Finally, activated monocytes responded to the addition of recombinant IL 2 by an increase in H2O2 production that was measured by using fluorescent indicator 2,7-dichlorofluorescein. This response as well as the observed induction of monocytic IL 2 receptors by LPS may point to a functional role for this receptor during monocyte/macrophage responses to microbial infections.

Cell Cycle↗
PubMed1987

Internalization of interleukin 2 is mediated by the beta chain of the high-affinity interleukin 2 receptor.

High-affinity IL-2-R correspond to a membrane receptor complex composed of two different IL-2-binding proteins, the Tac antigen (alpha chain) and a 70-75 kD beta chain. Using cell lines that express either the alpha or the beta protein, we demonstrate that IL-2 internalization occurs when ligand is bound to the isolated beta chain, but not when it is bound to the isolated alpha chain. The kinetics of IL-2 internalization mediated by the intermediate-affinity beta chain were nearly identical to those of the high-affinity alpha/beta heterodimer (t1/2 of 10-15 min), and each type of receptor targeted the bound IL-2 for intracellular degradation in lysosomes. The beta chain thus appeared to provide the essential element necessary for ligand internalization by both types of IL-2-R.

Animals↗