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.
Transcriptional activation of the human interleukin-2 (IL-2) gene, like induction of the IL-2 receptor alpha (IL-2R alpha) gene and the type 1 human immunodeficiency virus (HIV-1), is shown to be modulated by a kappa B-like enhancer element. Mutation of a kappa B core sequence identified in the IL-2 promoter (-206 to -195) partially inhibits both mitogen- and HTLV-I Tax-mediated activation of this transcription unit and blocks the specific binding of two inducible cellular factors. These kappa B-specific proteins (80 to 90 and 50 to 55 kilodaltons) similarly interact with the functional kappa B enhancer present in the IL-2R alpha promoter. These data suggest that these kappa B-specific proteins have a role in the coordinate regulation of this growth factor-growth factor receptor gene system that controls T cell proliferation.
We have investigated the biochemical basis for the activation of interleukin 2 receptor a-subunit gene expression in primary human T lymphocytes by a cytokine (tumor necrosis factor a), a T-cell mitogen (phorbol 12-myristate 13-acetate), and the transactivator protein (Tax) from the type I human T-cell leukemia virus. Using in vivo transfection techniques specificially designed for these primary T cells in coajunction with in vitro gel retardation and DNA footprinting assays, we found that activation of the IL-2Ra promoter by each of these agents involves the induction of nuclear proteins that specifically interact with a #cB-like enhancer element (i.e., an element resembling the immunoglobulin K-chain enhancer sequence recognized by transcription factor NF-iB). DNA-protein crosslinking studies revealed that primary T cells express at least three different inducible DNA-binding proteins (50)(51)(52)(53)(54)(55)(70)(71)(72)(73)(74)(75)(80)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90) that specifically interact with this IL-2Ra cB element.The proliferation of human T lymphocytes involves the coordinated activation of genes encoding the T-cell growth factor interleukin 2 (IL-2) and its membrane receptor (IL-2R) (1, 2). The functional high-affinity IL-2R complex consists of at least two distinct IL-2-binding protein subunits, IL-2Ra (Tac, p55, CD25) (3) and IL-2R1 (p70-75) (4-7). Both ofthese receptor subunits appear to be required for effective growthsignal transduction in the presence of physiological concentrations of 9). Since resting T cells constitutively express IL-2R3 (7), the induction of IL-2Ra gene expression is important for the regulation of high-affinity IL-2R display and T-cell proliferation. Cell surface expression of IL-2Ra can be induced by a variety of stimuli, including antigens, the transactivator protein (Tax) of type I human T-cell leukemia virus (HTLV-I) (10), and nonspecific mitogens such as phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) (11). In addition, tumor necrosis factor a (TNF-a), a macrophage-and T-cell-derived cytokine that displays a wide spectrum of biological activities (12), has been shown to induce IL-2Ra gene expression in both normal and leukemic T cells (13,14).Previous studies of the regulation of IL-2Ra gene activation have used long-term cultured leukemic T-cell lines as approximate experimental models for activation events occurring in primary T cells (15)(16)(17)(18)(19)(20)(21). Studies of one such T-cell line, Jurkat, suggested that the interaction of inducible DNA-binding proteins with a KB-like promoter element is important in the Tax activation of the IL-2Ra gene (19-21); however, disparate requirements for this element have been described with phorbol ester as the inducer (18-21).In the studies reported here, we analyzed the cis-acting regulatory sequences and trans-acting factors operational in the activation of IL-2Ra gene expression in primary human T cells induced with TNF-a, PMA, or Tax. Functional transfection studies employing a ...
Human im eficiency virus type 1 (HIV-1) is the causative agent of the acquired immunodeficiency syndrome (AIDS). Currently, no satisfactory treatment for this viral disease is available. Somatic gene therapy has been proposed as an alternative to conventional therapies. Several antiviral gene therapy approaches including ribozymes, antisense inhibition, and RNA-decoy stragW, well as dominant-negative mutants of HIV1pro*i (Qk, Tat, and Rev) have been suggested. To prove the concept Urans-dominant inhibition of HIV-1 replication, we transduced CEM cells with a retroviral vector encoding a dominant-negative rev gene.Amplification of integrase-specific proviral sequences from high molecular weight DNA indicated successfu HIV-1 human T-lymphotropic virus type fIB (HTLV-HIB) infection of all cells. In contrast to CEM cells and CEM cells expressing the rev wild-type (wt) gene, infection of two CEM-RevM1O clones with HIV-1 did not result in the release of significant levels of p24 Gag antigen as measured by antigen capture assay, indicating a block in HIV-1 replication due to the presence of the trans-dominant Rev protein. Furthermore, the parental CEM cells as well as CEM cells expressing the Rev wt protein were effectively killed in the course of the HIV-1 infection, whereas all CEM cells expressing the RevM10 protein were unaffected in their growth rate.The human immunodeficiency virus type 1 (HIV-1) is etiologically linked to AIDS (1, 2). HIV-1 is a complex human retrovirus encoding several regulatory genes (3). Specifically, the regulatory proteins Tat and Rev are crucially important for HIV-1 replication (4,5). The Rev transactivator protein interacts with a complex RNA structure, the Rev response element (RRE; refs. 6 and 7). This leads to the cytoplasmic accumulation of incompletely spliced HIV-1 transcripts, which are required for infectious particle formation. Therefore, the regulatory genes of HIV-1 represent targets for development of pharmacological antiviral therapies (8). In addition to antisense strategies (9), ribozymes (10,11), and dominant-negative Gag mutants (12), the HIV-1 regulatory proteins and their cis-acting RNA sequences are very attractive targets for somatic gene therapy approaches against AIDS. Trans-dominant Tat proteins have been described (13, 14) and overexpression of the trans-activation response element (TAR) RNA sequence in human T cells efficiently interfered with HIV-1 replication (15). The expression of chimeric neomycin-phosphotransferase (neo)-RRE transcripts was recently shown to interfere with Rev function in trans (16). Several dominant-negative mutants of the Rev trans-activator protein have been reported that suppress Rev wild-type (wt) function in transfection assays (17)(18)(19).In this study, we generated retroviral vectors that express the Rev wt protein as well as the dominant-negative RevM10 mutant protein (17) from an internal cytomegalovirus immediate early (CMV-IE) promoter. The retroviral DNAs were transfected into the GP+env Aml2 (Aml2) packaging lin...
Successful retroviral-mediated gene therapy will depend on safe, efficient packaging cell lines for vector particle production. Existing packaging lines for murine leukemia virus (MLV)-based vectors are predominantly derived from NIH/3T3 cells which carry endogenous MLV sequences that could participate in recombination to form replication-competent retrovirus (RCR). To identify cells devoid of such sequences, we screened genomic DNA from eight cell lines. DNA from the human 293 cell line did not cross-hybridize with MLV sequences, and these cells were able to secrete Gag particles after transfection. We derived a stable amphotropic packaging cell line (called ProPak-A) in 293 cells in which the Gag-Pol and Env (packaging) functions are expressed separately from a heterologous (non-MLV) promoter, to maximally reduce homology between packaging and vector sequences. ProPak-A-based producer cells are efficient, yielding higher stable titers than PA317-based producers. In addition, a vector that consistently gave rise to RCR in PA317 cells never resulted in detectable RCR in ProPak-A-based producer cultures. We have also shown that ProPak-A-packaged particles are not inactivated by human serum. Thus, the packaging cells we describe are as efficient and safer than the amphotropic packaging cells most commonly used in clinical gene therapy work and are also more appropriate for in vivo gene delivery.
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