HIV. Tampering with transcription.
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Animals↗
Biomedical subjects
G J Nabel
Publications and source records attributed to G J Nabel.
Expression of a novel nuclear protein in activated and in tat-I expressing T cells.
The intracellular events that occur in T lymphoid cells after activation or after infection with HIV-1 are not well defined. In the case of HIV-1 infection, it is unknown whether the tat-I gene, an essential gene for viral replication, affects host cell nuclear factors. Using two-dimensional PAGE, we have identified a novel nuclear protein, designated nuclear protein-28,000 (NP-28), which is induced in Jurkat T cells by stimulation with PMA and/or PHA or ionomycin. This nuclear protein has an apparent molecular mass of 28,000 Da and an isoelectric point of 4.6. Interestingly, Jurkat cells transfected with tat-I express higher levels of NP-28 constitutively, without added stimulation. Incubation of Jurkat cells expressing tat-I with PMA and/or PHA or ionomycin causes superinduction of NP-28. We have therefore identified a novel lymphoid nuclear protein induced by T cell activation that occurs in tat-I expressing cells in the absence of activating agents.
Genes, tat↗
Extrachromosomal human immunodeficiency virus type-1 DNA can initiate a spreading infection of HL-60 cells.
In this report, we describe a human immunodeficiency virus type-1 (HIV-1)-infected promyelocytic cell line, OM, derived from HL-60 cells. Although the OM cell line was biologically cloned twice, the pattern of HIV-1 expression during culture appeared analogous to a classical acute spreading infection and was inhibited by both azidothymidine and recombinant soluble CD4 treatment. The number of OM cells actually expressing HIV-1 at the beginning of culture was 0%, reached a peak of nearly 100% at 6 weeks, and then fell to less than 10% HIV-1+ cells by 10 weeks. Clonal analysis of the surviving cells verified that stable HIV-1+ OM cells resulted from the spreading infection. Southern analysis confirmed the transmission of HIV-1 through these OM cultures and the occurrence of stable clones which resulted. The initial percentage of OM cells actually harboring the HIV-1 genome was less than 0.1%, indicating nonfaithful transmission of an unintegrated HIV-1 genome during clonal expansion. These results demonstrate that extrachromosomal HIV-1 DNA can contribute to the spread of HIV-1 infection and give rise to cells which have stably integrated HIV-1 provirus.
Blotting, Southern↗
Gene transfer into vascular cells.
The goal of gene therapy is to introduce foreign deoxyribonucleic acid (DNA) into somatic cells to correct or prevent disorders caused by the malfunction of genes within a diseased individual. Overexpression of recombinant genes at specific sites within the vasculature can provide insights into vascular biology and potential treatments for various cardiovascular disorders such as restenosis. Methods for the introduction of foreign DNA into endothelial and vascular smooth muscle cells have been developed recently. These include the genetic modification of endothelium in vitro and implantation in vivo on arterial segments, direct infection of the arterial wall in vivo with a replication-defective retroviral vector expressing a recombinant gene and direct transfer of genes into vascular cells in vivo with use of liposomes. Although still in its formative stages, gene transfer into the vasculature holds promise as a potential treatment for vascular diseases, including atherosclerosis and restenosis. This approach may also provide insight into the role of specific gene products in the development of pathologic lesions.
Blood Vessel Prosthesis↗
Tumor necrosis factor-alpha, interleukin 1, and phorbol myristate acetate are independent activators of NF-kappa B which differentially activate T cells.
Gene expression in eukaryotic cells can be altered in different ways by extracellular agents, including mitogens and cytokines. Such differential gene expression is mediated in part through the effects of these stimuli on distinct sets of cellular transcription factors. In this report, the effects of phorbol myristate acetate, tumor necrosis factor-alpha (TNF-alpha), and interleukin 1 (IL-1) on differential gene expression in the LBRM mouse T-lymphoma cell line are examined. Although these three different stimuli produce similar levels of induction of the NF-kappa B transcription factor, it is reported that they cause differential expression of other cellular activation genes, including c-fos and IL-2. The roles of IL-1 and TNF-alpha were also analyzed in EL-4 cells in the presence of a second activator, ionomycin. IL-1, but not TNF-alpha, was found to stimulate the IL-2 enhancer in the presence of this costimulator. These findings suggest that one transcription factor can be the target of cellular activators that exert otherwise different effects on gene expression. Cellular activation pathways can therefore be defined by the set of transcription factors stimulated within a cell. This approach may allow a more precise definition of the requirements for differential gene activation in different cell types and thereby provide a basis for the selective manipulation of gene expression in cytokine-responsive cells.
Animals↗
Antisense oligonucleotides for therapeutic intervention.
Advances have been made in defining the best target sequences for use in antisense oligonucleotide technology, and new chemical derivatives of oligonucleotides are being investigated. Although the potential use of antisense oligonucleotide agents in the treatment of neoplastic, viral and parasitic diseases continues to be explored, they are not yet suitable for administration to humans for reasons that are discussed.
Animals↗
Oscillation of the human immunodeficiency virus surface receptor is regulated by the state of viral activation in a CD4+ cell model of chronic infection.
We have developed a unique physiologic model of chronic human immunodeficiency virus type 1 (HIV-1) infection, OM-10.1, clonally derived from infected HL-60 promyelocytes and harboring a single integrated provirus. Unlike other models of chronic infection, OM-10.1 cultures remain CD4+ under normal culture conditions, during which less than 10% of the cells constitutively express HIV-1 proteins. However, when treated with tumor necrosis factor alpha (TNF-alpha), OM-10.1 cultures dramatically increased (greater than 35-fold) HIV-1 expression and rapidly down-modulated surface CD4, as greater than 95% of the cells became HIV-1+. The complete loss of surface CD4 following viral activation was neither associated with apparent cytopathicity nor due to a decline of available CD4 mRNA. There was, however, a temporal association between CD4 down-modulation and the accumulation of intracellular HIV-1 gp 160/120; in addition, intracellular CD4-gp 160 complexes were identifiable in OM-10.1 cell lysates at time points following TNF-alpha induction after surface CD4 was no longer detectable. Surface CD4 expression by OM-10.1 cells returned once viral activation ceased and could be repeatedly oscillated upon HIV-1 reactivation. Furthermore, inhibition of protein kinase activity following maximal TNF-alpha stimulation of OM-10.1 cells quickly returned activated HIV-1 to a state of latency, as evidenced by an accelerated return of surface CD4. These results with the new OM-10.1 cell line demonstrate that CD4 surface expression can be maintained during chronic infection and is critically dependent on the state of viral activation, that CD4-gp 160 intracellular complexing is involved in CD4 down-modulation, and that protein kinase pathways not only function in the primary induction of latent HIV-1 but also are required for maintaining the state of viral activation.
Blotting, Southern↗
Introduction of vascular smooth muscle cells expressing recombinant genes in vivo.
Vascular smooth muscle cells contribute to the formation of atherosclerotic plaques by proliferating in response to vascular injury and releasing growth-promoting factors. Because their autocrine and paracrine effects are not fully understood, expression of such growth factor genes in specific cell types in vivo would help to determine their mechanism of action. We describe a method to transfer vascular smooth muscle cells expressing recombinant gene products to localized segments of the arterial wall. Vascular smooth muscle cells from the inbred Yucatan minipig were infected in vitro with an amphotropic, replication-defective retrovirus transducing the gene for Escherichia coli beta-galactosidase. Vascular smooth muscle cells expressing this recombinant gene were implanted, using a catheter, into denuded iliofemoral artery segments of pigs in vivo. These arteries subsequently demonstrated beta-galactosidase activity in cells of the intima and media. This method, which provides for the introduction of genetically modified smooth muscle cells, can be used to define the biological effects of recombinant genes in the vessel wall and potentially to provide alternative treatments of vascular diseases.
Animals↗
Selective elimination of recombinant genes in vivo with a suicide retroviral vector.
The ability to express recombinant genes in vivo offers potential new treatments for human disease if questions of safety and toxicity can be addressed. Complications of gene transfer could include, for example, overexpression of introduced genes for growth or angiogenic factors or insertional mutagenesis, both of which could cause uncontrolled cell growth. We report the development of a suicide retroviral vector that provides a method to eliminate cells undergoing rapid growth in vivo. A murine amphotropic retroviral vector was constructed in which the gene for herpesvirus thymidine kinase was included to render proliferating cells sensitive to ganciclovir, and the Escherichia coli beta-galactosidase gene served as a reporter. This vector's efficacy was first assessed in vitro, and beta-galactosidase activity was abolished in several cell lines after treatment with ganciclovir. In vivo, a transplantable murine CT26 adenocarcinoma whose cells were transduced with this vector regressed completely after administration of ganciclovir. In contrast, expression in nondividing cells within rabbit arteries transduced by retroviral infection in vivo was unaffected. This suicide vector therefore eliminates transformed cells but allows survival of normal nondividing cells that express its specific recombinant genes in vivo, and may thus improve the safety and efficacy of gene transfer into living organisms.
Adenocarcinoma↗
Cloning of R kappa B, a novel DNA-binding protein that recognizes the interleukin-2 receptor alpha chain kappa B site.
Transcriptional activation of the interleukin-2 receptor alpha (IL-2R alpha) gene in T cells is dependent on a regulatory element that can bind NF-kappa B but differs in sequence and function from the kappa B site of the immunoglobulin (Ig) enhancer. To define the molecular basis of gene-specific regulation by this variant kappa B site, we have used electrophoretic mobility shift assays to characterize a novel gene product, designated R kappa B, that binds preferentially to the related kappa B site in IL-2R alpha. A cDNA encoding this 107-kD protein has been isolated from a lambda gt11 expression library by screening with a probe containing the IL-2R alpha kappa B site. R kappa B is a tissue-specific transcription factor that contains an amino acid sequence similar to a discrete region of the myogenic regulatory protein MyoD, but is unrelated to other DNA-binding proteins, including those belonging to the rel/dorsal gene family. This novel kappa B binding protein may therefore contribute to the regulation of distinct cellular and viral genes with variant kappa B sites.
Amino Acid Sequence↗
Regulation of gene expression with double-stranded phosphorothioate oligonucleotides.
Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins is necessary for determining how these factors participate in cellular differentiation. The functions of these proteins can be antagonized by several methods, each with specific limitations. Inhibition of sequence-specific DNA-binding proteins was achieved with double-stranded (ds) phosphorothioate oligonucleotides that contained octamer or kappa B consensus sequences. The phosphorothioate oligonucleotides specifically bound either octamer transcription factor or nuclear factor (NF)-kappa B. The modified oligonucleotides accumulated in cells more effectively than standard ds oligonucleotides and modulated gene expression in a specific manner. Octamer-dependent activation of a reporter plasmid or NF-kappa B-dependent activation of the human immunodeficiency virus (HIV) enhancer was inhibited when the appropriate phosphorothioate oligonucleotide was added to a transiently transfected B cell line. Addition of phosphorothioate oligonucleotides that contained the octamer consensus to Jurkat T leukemia cells inhibited interleukin-2 (IL-2) secretion to a degree similar to that observed with a mutated octamer site in the IL-2 enhancer. The ds phosphorothioate oligonucleotides probably compete for binding of specific transcription factors and may provide anti-viral, immunosuppressive, or other therapeutic effects.
DNA-Binding Proteins↗
Site-specific gene expression in vivo by direct gene transfer into the arterial wall.
A recombinant beta-galactosidase gene has been expressed in a specific arterial segment in vivo by direct infection with a murine amphotropic retroviral vector or by DNA transfection with the use of liposomes. Several cell types in the vessel wall were transduced, including endothelial and vascular smooth muscle cells. After retroviral infection, a recombinant reporter gene was expressed for at least 5 months, and no helper virus was detected. Recombinant gene expression achieved by direct retroviral infection or liposome-mediated DNA transfection was limited to the site of infection and was absent from liver, lung, kidney, and spleen. These results demonstrate that site-specific gene expression can be achieved by direct gene transfer in vivo and could be applied to the treatment of such human diseases as atherosclerosis or cancer.
Actins↗
Differential regulation of human immunodeficiency viruses (HIVs): a specific regulatory element in HIV-2 responds to stimulation of the T-cell antigen receptor.
The human immunodeficiency viruses (HIVs) types 1 and 2 have similar genetic organization but differ significantly in nucleic acid sequence. Although infection by either agent leads to symptoms of immunodeficiency, recent studies suggest potential differences in the time course and severity of these diseases. In this report, the transcriptional regulation and induction of these retroviruses were analyzed. We report that the regulation of HIV-2 differs from that of HIV-1: a distinct T-cell activation pathway, triggering of the CD3 component of the T-cell receptor complex, stimulates HIV-2 but not HIV-1 gene expression. The response to T-cell receptor stimulation in HIV-2 is mediated partly by an upstream regulatory element, termed CD3R, which is recognized by a sequence-specific DNA binding protein, NF-CD3R. Jurkat T leukemia cell lines containing HIV-2 provirus also showed increased viral replication after stimulation of the T-cell receptor complex, in contrast to HIV-1. These findings suggest that transcriptional regulation and induction of HIV-2 differ from HIV-1 and raise the possibility that different cofactors contribute to the activation of HIV-1- and HIV-2-associated AIDS.
Antigens, CD↗
Regulation of the IL-2 receptor alpha-gene. Interaction of a kappa B binding protein with cell-specific transcription factors.
The IL-2R alpha enhancer contains an 11 bp sequence that resembles kappa B, a regulatory element associated with several genes, including Ig kappa-L chain and human immunodeficiency virus. Although nuclear factor of the kappa-enhancer in B cells (NF-kappa B) binding is activated by PMA, TNF-alpha, and IL-1, activation of the IL-2R alpha enhancer does not consistently correlate with NF-kappa B induction. In this report, we show that TNF-alpha activates NF-kappa B and the human immunodeficiency virus enhancer in the Jurkat T leukemia but does not stimulate the IL-2R alpha enhancer. In contrast, this cytokine, and IL-1, increased IL-2R alpha gene expression in YT-1 cells. Comparing YT-1 and Jurkat T leukemias, we find that the IL-2R kappa B site is required for TNF-alpha and IL-1 stimulation in YT-1 cells, but that plasmids containing this site linked to a heterologous promoter do not respond to these cytokines. These data suggest that upstream regulatory elements in addition to IL-2R kappa B are needed to mediate this cytokine effect. TNF-alpha also synergized with PMA and other cytokines in the stimulation of the IL-2R alpha enhancer in YT-1. Because these effects are not observed in Jurkat cells, the function of the IL-2R kappa B site is cell-specific and likely mediated by different associated transcription factors present in each cell type.
DNA-Binding Proteins↗
Recombinant gene expression in vivo within endothelial cells of the arterial wall.
A technique for the transfer of endothelial cells and expression of recombinant genes in vivo could allow the introduction of proteins of therapeutic value in the management of cardiovascular diseases. Porcine endothelial cells expressing recombinant beta-galactosidase from a murine amphotropic retroviral vector were introduced with a catheter into denuded iliofemoral arteries of syngeneic animals. Arterial segments explanted 2 to 4 weeks later contained endothelial cells expressing beta-galactosidase, an indication that they were successfully implanted on the vessel wall.
Animals↗
Activation of HIV gene expression during monocyte differentiation by induction of NF-kappa B.
The latent period of AIDS is influenced by factors which activate human immunodeficiency virus (HIV) replication in different cell types. Although monocytic cells may provide a reservoir for virus production in vivo, their regulation of HIV transcription has not been defined. We now report that HIV gene expression in the monocyte lineage is regulated by NF-kappa B, the same transcription factor known to stimulate the HIV enhancer in activated T cells; however, control of NF-kappa B and HIV in monocytes differs from that observed in T cells. NF-kappa B-binding activity appears during the transition from promonocyte to monocyte in U937 cells induced to differentiate in vitro and is present constitutively in mature monocytes and macrophages. In a chronically infected promonocytic cell, U1, differentiation is associated with HIV-1 replication as well as NF-kappa B binding activity. These findings suggest that NF-kappa B binding activity is developmentally regulated in the monocyte lineage, and that it provides one signal for HIV activation in these cells.
Cell Differentiation↗
Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B.
Binding of peptide hormones to surface membrane receptors leads to the transcription of specific genes within relevant target cells. How these signals are transduced to alter gene expression is largely unknown, but this mechanism probably involves a sequence of enzymatic steps that activate factors in the nucleus that modulate transcription. We now demonstrate that two different peptide hormones, or cytokines, stimulate the human immunodeficiency virus enhancer, and this effect is mediated by nuclear factor (NF) kappa B (nuclear factor that binds the kappa immunoglobulin light chain gene enhancer). These cytokines, tumor necrosis factor alpha and interleukin 1, act on multiple cell types and represent the only naturally occurring activators of this transcription factor among eight cytokines examined. Although NF-kappa B binding can be stimulated by phorbol 12-myristate 13-acetate, tumor necrosis factor alpha acts through an independent mechanism, inducing NF-kappa B binding in HT-2 cells, which did not show increased binding in response to phorbol 12-myristate 13-acetate, and causing superinduction in Jurkat T-lymphoma cells. Tumor necrosis factor alpha is also a more selective activator of T cells than phorbol 12-myristate 13-acetate, having no effect on lymphokine production in EL-4 cells at the same time it induces NF-kappa B. These findings suggest that human immunodeficiency virus gene expression can be induced in T cells without activating lymphokine secretion and that the role of these cytokines in the activation of latent human immunodeficiency virus infection deserves further clinical evaluation. Finally, this link between binding at the surface membrane and stimulation of a specific transcription factor should help define intermediates for these cytokine activation pathways.
Cell Line↗
NF-kappa B-mediated activation of the human immunodeficiency virus enhancer: site of transcriptional initiation is independent of the TATA box.
The activation of the human immunodeficiency virus (HIV) enhancer in T cells can occur through multiple independent pathways. This enhancer is stimulated by NF-kappa B or through alternative mechanisms, including E1A transactivation, which is dependent on the TATA box. In this report, the role of the TATA box in activation by NF-kappa B is examined. E1A stimulation of the HIV enhancer requires the presence of the TATA sequence in the sense orientation. Analysis of mutant HIV enhancer plasmids shows that basal mRNA levels are reduced when the TATA sequence is altered but that inducibility of NF-kappa B and the site of transcriptional initiation are unchanged. These data suggest that transcriptional initiation in this class II promoter is determined by an initiator factor which does not require binding to the TATA sequence. Because kappa B is found in a variety of viruses, this mechanism may be relevant to the activation of other viral enhancers.
Adenovirus Early Proteins↗