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PubMed1998

p53 transactivation of the HIV-1 long terminal repeat is blocked by PD 144795, a calcineurin-inhibitor with anti-HIV properties.

Previous reports have indicated that benzothiophenes exhibit broad anti-inflammatory properties and inhibit human immunodeficiency virus-type 1 (HIV-1) replication. We show that the immunosuppressant cyclosporin A (CsA) and benzothiophene-2-carboxamide, 5-methoxy-3-(1-methyl ethoxy)-1-oxide (PD 144795) block the induction of p53 and NF-kappaB binding to the HIV-1 long terminal repeat (LTR) by the T cell receptor activator phytohemagglutinin. CsA and PD 144795 also inhibit the induction by phytohemagglutinin of the transcription mediated by an HIV-1 LTR fragment containing the p53 and NF-kappaB sites. These effects of PD 144795 on HIV-1 transcription correlate with its ability to inhibit the phosphatase activity of calcineurin and are similar to those previously described for CsA. Moreover, a constitutive active form of calcineurin is able to induce expression from the HIV-1 LTR in a p53- and NF-kappaB-dependent manner and PD 144795 is able to block this induction. These results demonstrate that the DNA binding of p53 to the HIV-1 LTR can be modulated by calcineurin and provide a framework to understand the anti-HIV properties of benzothiophene derivatives.

Anti-HIV Agents↗
PubMed1998

Hematopoietic transcription factor GATA-2 activates transcription from HIV-1 long terminal repeat.

OBJECTIVES: To study the role of the hematopoietic transcription factor GATA-2 in long terminal repeat (LTR)-directed transcriptional activation of HIV-1 in hematopoietic progenitor cells, and to investigate possible GATA-2 binding sites in HIV-1 LTR. DESIGN AND METHODS: Wild-type HIV-1 LTR, or mutants, ligated to a luciferase reporter gene with or without a GATA-2 expression vector, were transfected into COS cells, and standardized luciferase activity was examined. The binding activity of GATA-2 to these sites was examined by electrophoretic mobility shift assay. These wild-type or mutant reporter genes were also transfected into the murine hematopoietic progenitor cells, BAF3, in which GATA-2 was the predominantly expressed transcription factor of the GATA family, to assay LTR-directed transcription in intact hematopoietic machinery. Using a Tat expression plasmid for cotransfection, the influence of Tat protein on GATA-2-induced transactivation was determined. RESULTS: In COS cells, LTR-dependent transactivation was highly enhanced by the coexpression of GATA-2. Experiments with mutant LTR suggested the presence of multiple GATA-2 binding sites, of which the major sites were identified. Cotransfection of Tat with GATA-2 indicated that GATA-2 and Tat synergistically enhanced the transcriptional activity. Transfection experiments in BAF3 cells showed that the disruption of these GATA sites diminished LTR-driven activity to 40% of the wild-type. CONCLUSIONS: GATA-2 may be a key host cell regulator of HIV-1 expression in hematopoietic stem cells. Manipulating this transactivation may represent a valuable approach to controlling virus production in infected hematopoietic progenitors. To elucidate the possible interaction between GATA-2 and Tat protein in vivo might give new insights to the mechanism of impaired hematopoiesis in AIDS patients.

Animals↗
PubMed1998

Human cytomegalovirus product UL44 downregulates the transactivation of HIV-1 long terminal repeat.

OBJECTIVE: Human cytomegalovirus (HCMV) is often isolated from HIV-1-infected patients and the two viruses can infect the same cell type giving rise to direct bidirectional interactions. Whereas the long terminal repeat (LTR) transactivation ability of HCMV immediate early gene (IE1/IE2) is well documented, no information is available on the possible role of other HCMV proteins. In this study, the activity of ppUL44, an early DNA-binding protein, on HIV LTR transactivation was investigated. METHODS: HIV LTR transactivation by ppUL44 in presence or absence of HIV-1 Tat and HCMV IE1/IE2 was determined in J-Jhan and U973 cells through transient transfection experiments with a series of different expression vectors. Some experiments were also performed on U373-MG astrocytoma cells permanently transfected with UL44 or with another HCMV gene used as a control (UL55). RESULTS: The basal transactivation activity of the HIV LTR was not influenced by the presence of ppUL44. On the contrary, the transactivation observed in the presence of Tat, IE1/IE2 or both factors in synergy was strongly downregulated by ppUL44 in a dose-dependent manner. Deletion constructs of ppUL44 demonstrated that the region of the molecule responsible for the inhibition of the LTR is located within the last 114 amino acids at the carboxyl-terminal region. CONCLUSIONS: The results obtained indicate that within the last 114 amino acids of ppUL44 there is a domain that has a negative effect on the ability of HIV-1 LTR to be activated by both its autologous transactivator Tat and the heterologous transactivator HCMV IE1/IE2 functioning individually or synergistically.

Astrocytoma↗
PubMed1993

Effects of integration and replication on transcription of the HIV-1 long terminal repeat.

The activity of a promoter is influenced by chromosomal and cell cycle/replication context. We analyzed the influences of integration and replication on transcription of the human immunodeficiency virus (HIV)-1 long terminal repeat (LTR). We found that one requirement for Tat trans-activated expression differed for integrated versus unintegrated HIV-1 LTR. Specifically, the second coding exon of Tat, previously regarded as functionally dispensable, plays a role in the optimal activation of integrated LTRs. We also found that the transcription profile produced from the HIV-1 LTR is influenced by replication. Autonomously replicating vectors that contain the HIV-1 LTR produced patterns of transcription different from counterpart vectors that do not replicate. Both replicating and nonreplicating HIV-1 LTRs remained responsive to Tat.

Antigens, Polyomavirus Transforming↗
PubMed2001

The hepatitis B virus X protein induces HIV-1 replication and transcription in synergy with T-cell activation signals: functional roles of NF-kappaB/NF-AT and SP1-binding sites in the HIV-1 long terminal repeat promoter.

Co-infection with hepatitis B virus (HBV) and human immunodeficiency virus type-1 (HIV-1) is relatively common. However, the impact of this co-infection on the clinical outcome of HIV infection has not been elucidated. We herein demonstrate that the HBV X protein (HBx) superinduces ongoing HIV-1 replication and HIV-1 long terminal repeat (LTR) transcription by synergizing with Tat protein and with T-cell activation signals. Although HBx cooperated with mitogenic stimuli in the induction of reporter plasmids harboring the HIV-1 kappaB enhancer, in both a NF-kappaB-dependent manner and a NF-AT-dependent manner, deletion of this element from the LTR did not affect the HBx-mediated up-regulation in the presence of Tat and/or mitogens. In contrast, mutation of the proximal LTR Sp1-binding sites abolished the HBx-mediated synergistic activation, but only when it was accompanied by deletion of the kappaB enhancer. When HBx was targeted to the nucleus, its ability to synergize with cellular activation stimuli was maintained. Furthermore, mutations of HBx affecting its interaction with the basal transcription machinery abrogated the synergistic activation by HBx, suggesting that this protein exerts its function by acting as a nuclear co-activator. These results indicate that HBx could contribute to a faster progression to AIDS in HBV-HIV co-infected individuals.

Base Sequence↗
PubMed1991

Cloning of a cellular factor, interleukin binding factor, that binds to NFAT-like motifs in the human immunodeficiency virus long terminal repeat.

Human immunodeficiency virus (HIV) gene expression is regulated by both general transcription factors and factors induced by activation of T lymphocytes such as NF-kappa B and the nuclear factor of activated T cells (NFAT). Within the HIV long terminal repeat (LTR), two purine-rich domains between nucleotides -283 and -195 have homology to a regulatory region found in the interleukin 2 promoter, which binds NFAT and other cellular factors. In the HIV LTR, this region has been demonstrated to have both positive and negative regulatory effects on HIV gene expression. In an attempt to clone genes encoding cellular factors that bind to these NFAT-like elements in the HIV LTR, we used lambda gt11 expression cloning with oligonucleotides corresponding to these binding motifs. A ubiquitously expressed cDNA encoding a 60-kDa protein, which we termed interleukin binding factor (ILF), binds specifically to these purine-rich motifs in the HIV LTR. This factor also binds to similar purine-rich motifs in the interleukin 2 promoter, through with lower affinity than to HIV LTR sequences. Sequence analysis reveals that the DNA binding domain of ILF has strong homology to the recently described fork head DNA binding domain found in the Drosophila homeotic protein fork head and a family of hepatocyte nuclear factors, HNF-3. Other domains found in ILF include a nucleotide binding site, an N-glycosylation motif, a signal for ubiquitin-mediated degradation, and a potential nuclear localization signal. These results describe a DNA binding protein that may be involved in both positive and negative regulation of important viral and cellular promoter elements.

Amino Acid Sequence↗
PubMed1997

Activation of the HIV-1 long terminal repeat by nerve growth factor.

The brain is an important target for the human immunodeficiency virus type 1 (HIV-1). We show here that nerve growth factor (NGF), which induces neuronal differentiation and survival, causes a strong activation of the HIV-1 long terminal repeat by a Ras/Raf-dependent mechanism in PC12 cells. Mutation of the kappaB sequences contained whithin the long terminal repeat reduces NGF-mediated stimulation. NGF does not activate NF-kappaB in PC12 cells, but rather increases binding of other nuclear factors to the kappaB sequences. Furthermore, a nuclear receptor response element contributes to the stimulatory effect of NGF. The retinoids receptors have been identified as components of the nuclear binding to the nuclear receptor response element in NGF-treated PC12 cells. These results reveal the importance of neurotrophins and nuclear receptor signaling pathways as specific activators of HIV-1 gene expression in neural cells.

Animals↗
PubMed1996

Activation of the HIV-1 long terminal repeat by cytokines and environmental stress requires an active CSBP/p38 MAP kinase.

The human immunodeficiency virus, type 1 (HIV-1) promoter is known to be activated by proinflammatory cytokines and UV light. These stimuli also activate various members of the mitogen-activated protein kinase family, including JNK/SAPK and CSBP/p38. In HeLa cells containing an integrated HIV-1 long terminal repeat (LTR) -driven reporter, we now show that the specific p38 inhibitor, SB203580, inhibits activation of the HIV-1 LTR by interleukin-1, tumor necrosis factor, UV light, and osmotic stress. Inhibition was 70-90% in all but the case of tumor necrosis factor stimulation, where inhibition was 50%. Each of these stimuli activated p38, which was inhibited by SB203580 in vitro and in vivo with an IC50 (between 0.1 and 1 microM) similar to that required to inhibit transcription. In contrast, SB203580 had no effect on JNK, which was also activated by these stimuli. The NFkappaB sites in the HIV-1 LTR were required for a response to cytokines but not to UV, and SB203580 remained capable of inhibiting UV activation in the absence of the NFkappaB sites. Studies in which SB203580 was added at different times relative to UV stimulation suggested that the critical p38-mediated phosphorylation event occurred between 2 and 4 h after UV treatment. These data indicate that p38 is required for HIV-1 LTR activation but that the action of p38 is delayed, presumably due to substrate unavailability or inaccessibility.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1989

UV-induced transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat and UV-induced secretion of an extracellular factor that induces HIV-1 transcription in nonirradiated cells.

UV irradiation, but not visible sunlight, induces the transcription of human immunodeficiency virus type 1 (HIV-1). Chimeric constructs carrying all or parts of the HIV-1 long terminal repeat linked to an indicator gene were transfected into HeLa cells or murine and human T-cell lines, and their response to irradiation was tested. The cis-acting element conferring UV responsiveness is identical to the sequence binding transcription factor NF kappa B. UV irradiation enhances NF kappa B binding activity as assayed by gel retardation experiments. Interestingly, the requirement for UV irradiation can be replaced by cocultivation of transfected cells with UV-irradiated nontransfected (HIV-1-negative) cells. A UV-induced extracellular protein factor is detected in the culture medium conditioned by UV-treated cells. The factor is produced upon UV irradiation by several murine and human cell lines, including HeLa, Molt-4, and Jurkat, and acts on several cells. These data suggest that the UV response of keratinocytes in human skin can be magnified and spread to deeper layers that are more shielded, including the Langerhans cells, and that this indirect UV response may contribute to the activation of HIV-1 in humans.

Cell Line↗
PubMed1995

Mycoplasmas regulate HIV-LTR-dependent gene expression.

Mycoplasmas have been incriminated in setting the stage for HIV infection and full-blown AIDS. We tested the possible involvement of mycoplasmas in activation of HIV. Two cell lines, 293 fibroblasts and Jurkat CD4+ T-cells, transfected with plasmids harboring a transcription fusion construct between HIV-long terminal repeat (HIV-LTR) and either luc or cat genes, were infected with several mycoplasmas (M. fermentans; M. penetrans, M. pirum and Ureaplasma urealyticum) and the reporter gene expression was monitored. The data presented here suggest that mycoplasmas, and specifically their membranes, play a role in the activation of HIV-LTR mediated transcription.

CD4-Positive T-Lymphocytes↗
PubMed1992

Epstein-Barr virus latent membrane protein transactivates the human immunodeficiency virus type 1 long terminal repeat through induction of NF-kappa B activity.

The Epstein-Barr virus latent membrane protein (LMP) is an integral membrane protein that is expressed in cells latently infected with the virus. LMP is believed to play an important role in Epstein-Barr virus transformation and has been shown to induce expression of several cellular proteins. We performed a series of experiments that demonstrated that LMP is an efficient transactivator of expression from the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). Mutation or deletion of the NF-kappa B elements in the LTR abolished the transactivation, indicating that the LMP effect on HIV expression was due to induction of NF-kappa B activity. Experiments in which the HIV-1 Tat protein was coexpressed in cells together with LMP showed that Tat was able to potentiate the transactivation. Surprisingly, a synergistic effect of the two proteins was observed even in the absence of the recognized target region for Tat (TAR) in the HIV-1 LTR.

Burkitt Lymphoma↗
PubMed1994

Sequence-specific recognition of the HIV-1 long terminal repeat by distamycin: a DNAase I footprinting study.

Pharmacological modulation of the interaction between transcription factors and target DNA sequences of cellular and viral genes could have important effects in the experimental therapy of a large variety of human pathologies. For instance, alteration of the DNA/protein interaction might be among the molecular mechanisms of action of DNA-binding drugs, leading to an inhibition of the expression of genes involved in the control of in vitro and in vivo growth of neoplastic cells and virus DNA replication. Natural oligopeptides, such as distamycin, are powerful inhibitors of the interaction between nuclear factors and target DNA sequences and, therefore, have been proposed as compounds retaining antibiotic, antineoplastic and antiviral properties. In this study we performed DNAase I footprinting analysis using a PCR product mimicking a region of the long terminal repeat (LTR) of the human immunodeficiency type 1 (HIV-1) retrovirus. The data obtained suggest that distamycin binds to different regions of the HIV-1 LTR depending on the DNA sequence. Electrophoretic mobility shift assays using both crude nuclear extracts from the Jurkat T-lymphoid cell line and the recombinant proteins transcription factor IID and Sp1 suggest that distamycin differentially inhibits the interaction of these two proteins with their specific DNA target sequences, in good agreement with the results obtained by DNAase I footprinting analysis.

Base Sequence↗
PubMed1999

Dual activity of pyrrolidine dithiocarbamate on kappa B-dependent gene expression in U937 cells: I. Regulation by the phorbol ester TPA.

Pyrrolidine dithiocarbamate (PDTC) has been widely used as an inhibitor of the nuclear factor-kappa B, (NF-kappa B) signalling pathway. Here, we show that kappa B-dependent reporter gene expression induced by low concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) is potentiated by PDTC in the human pro-monocytic U937 cell line. The stimulatory effect of PDTC on kappa B-dependent gene expression was shown with a 4 x kappa B chloramphenicol acetyltransferase construct and required an intact kappa B element in the human immunodeficiency virus long terminal repeat (HIV-1 LTR). Unexpectedly, an HIV-1 LTR construct with a mutation of the activator protein 2 (AP-2) binding site located between the two kappa B elements was unresponsive to the stimulatory effect of PDTC with TPA. The stimulation or inhibition of kappa B-dependent gene expression was dependent on PDTC pre-treatment and the concentration of TPA. No stimulatory effect on HIV-1 LTR activity was observed with the metal chelator dipyridyl or the anti-oxidant N-acetyl-L-cysteine. These results are consistent with the hypothesis that PDTC treatment potentiated kappa B-dependent gene expression in a manner dependent on the concentration of TPA.

Chelating Agents↗
PubMedDate not supplied

Hexamethylene bisacetamide and cis-platin stimulate the expression from the HIV-1 long terminal repeat sequence in human MCF-7 cells.

We have employed a recombinant plasmid pBHIV-1 carrying the Long Terminal Repeat sequences (LTR) of the Human Immunodeficiency Virus (HIV-1) linked to the reporter chloramphenicol acetyl transferase (CAT) gene and to the aminoglycoside phosphotransferase (aph) gene as a selectable marker. We have introduced pBHIV-1 DNA in human breast cancer MCF-7 cells and obtained the MCF-7HIV-1 cell line, resistant to geneticin. We have studied the effect of hexamethylene-bisacetamide (HMBA), cis-platin, interferon-aB, dexamethasone and tamoxifen on the LTR regulated CAT activity in MCF-7HIV-1 cells. It was found that HMBA and cis-platin stimulated CAT activity, whereas interferon-aB, dexamethasone and tamoxifen had no significant effect.

Acetamides↗
PubMed1993

Activation of the human immunodeficiency virus type 1 long terminal repeat by BTEB, a GC box-binding transcription factor.

BTEB, a GC box-binding transcription factor, was tested for its ability to activate the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). An electrophoretic mobility shift assay demonstrated specific binding of BTEB to GC boxes of the HIV-1 LTR. When a BTEB expression vector was cotransfected into A3.01 cells with a fusion gene of HIV-1 LTR and chloramphenicol acetyltransferase (CAT) structural gene, the CAT activity was increased. This increase was accompanied by an increase in the content of CAT mRNA. Transcriptional activity of the HIV-1 LTR, stimulated by Tat, was further enhanced by the expression of BTEB. BTEB also activated the LTR activity in cooperation with phorbol 12-myristate 13-acetate. Northern blot analysis showed that various T cell and macrophage/monocyte cell lines expressed the BTEB mRNA to a level comparable with that of Sp1, another GC box-binding transcription factor. These results suggest that BTEB, like Sp1, is involved in transcriptional activation of the HIV-1 LTR.

Base Sequence↗
PubMed1996

Tyrosine kinase and cAMP-dependent protein kinase activities in CD40-activated human B lymphocytes.

In vitro, human B lymphocytes undergo long-term proliferation when activated through CD40, a protein expressed on their cell surface. The nature of CD40-dependent signals in proliferating fresh human Epstein-Barr virus-negative B lymphocytes is currently unknown. In this study, a CD40-dependent B cell culture system was used to examine the role of different signal transduction elements. Protein kinase C (PKC) depletion generated by a long-term phorbol 12 myristate 13-acetate treatment had weak effects on proliferation. Rather, tyrosine phosphorylation was shown to be directly involved in mediating CD40-dependent signals. The use of the protein tyrosine kinase (PTK)-specific inhibitor herbimycin A dramatically decreased cellular proliferation without altering the activity of the human immunodeficiency virus-1 long terminal repeat (HIV-1 LTR), a promoter largely dependent on the binding of nuclear factor kappa B (NF- kappa B). In contrast, the cAMP-dependent protein kinase specific inhibitor H-89 totally inhibited HIV-1 LTR activity at a concentration as low as 100 nM without affecting cellular proliferation. Electrophoretic mobility shift assay (EMSA) and supershift assay using an NF-kappa B binding sequence from the kappa light chain as a probe, revealed that both p65 (RelA) and c-Rel were present in CD40-stimulated B cells. While PKC depletion did not alter the NF-kappa B level, treatment of B lymphocytes with H-89 or herbimycin A provoked a decrease in the NF-kappa B level. These observations establish the importance of different signal transducing pathways leading to CD40 activation of B lymphocytes.

B-Lymphocytes↗
PubMed1992

Human chromosome 12 is required for optimal interactions between Tat and TAR of human immunodeficiency virus type 1 in rodent cells.

Levels of trans activation of the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) by the virally encoded transactivator Tat show marked species-specific differences. For example, levels of transactivation observed in Chinese hamster ovary (CHO) rodent cells are 10-fold lower than those in human cells or in CHO cells that contain the human chromosome 12. Thus, the human chromosome 12 codes for a protein or proteins that are required for optimal Tat activity. Here, the function of these cellular proteins was analyzed by using a number of modified HIV-1 LTRs and Tats. Neither DNA-binding proteins that bind to the HIV-1 LTR nor proteins that interact with the activation domain of Tat could be implicated in this defect. However, since species-specific differences were no longer observed with hybrid proteins that contain the activation domain of Tat fused to heterologous RNA-binding proteins, optimal interactions between Tat and the trans-acting responsive RNA (TAR) must depend on this factor(s).

Animals↗
PubMed1993

Differential interaction of cisplatin with the HIV-1 long terminal repeat in a resistant ovarian carcinoma cell line.

We constructed a recombinant plasmid, pBHIV1, carrying the long terminal repeat (LTR) sequences of HIV-1 linked to the reporter chloramphenicol acetyl transferase (CAT) gene and to the aminoglycoside phosphotransferase (aph) gene as a selectable marker. We have introduced pBHIV1 in a human ovarian cancer cell line A2780 and in a cisplatin resistant variant 2780CP, and obtained stable geneticin resistant A27HIV1-1 and 27CPHIV1-1 cells, respectively. Both transfectant cells expressed CAT activity from the HIV LTR promoter. The response to the anti-neoplastic drug cisplatin was studied on the LTR regulated CAT activity in both cell lines. It was found that cisplatin at 2.5 x 10(-5) M concentration stimulates the expression of CAT by 26-fold from the HIV LTR in A27HIV1-1, but requires a concentration of 5 x 10(-5) M to enhance expression by 4.1-fold in the cisplatin resistant 27CPHIV1-1 cells. Carboplatin, over a range of concentrations (1 x 10(-6) to 1 x 10(-4) M), does not stimulate expression of CAT from the HIV-1 LTR in either of the transfected cells.

Carboplatin↗