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At least 19 recordsLinked to original sources
PubMed1991

Anti-CD2 receptor antibodies activate the HIV long terminal repeat in T lymphocytes.

The CD2 T lymphocyte glycoprotein surface molecule mediates both cell to cell adhesion and T cell activation, two processes that are involved in the spread of HIV infection. Treatment of chronically HIV-infected PBMC with anti-CD2 mAb has been shown to induce the expression of infectious virus from these cultures. In this study we investigated the mechanisms whereby anti-CD2 antibodies stimulate viral production. We demonstrate that treatment of transiently transfected T lymphocytes with anti-CD2 antibodies results in activation of the HIV long terminal repeat. Furthermore, CAT assays using mutated HIV long terminal repeat-CAT constructs and gel shift assays demonstrate that this activation is dependent on the NF-kappa B enhancer. These studies suggest that interaction of CD2 with its natural ligand, LFA-3, may play a role in regulation of HIV expression.

Antibodies, Monoclonal↗
PubMed1990

Constitutive expression of human immunodeficiency virus (HIV) nef protein in human astrocytes does not influence basal or induced HIV long terminal repeat activity.

Since human immunodeficiency virus (HIV) nef has been suggested to exert regulatory effects on HIV long terminal repeat (LTR) activity, we transiently transfected HIV LTR chloramphenicol acetyltransferase or luciferase expression vectors into a human astrocytoma clone (U-373nef) that constitutively expresses the HIV nef gene. In these cells, basal HIV LTR activity, as well as tumor necrosis factor-induced or tat-driven activity, was similar to that in control cells. Lack of any detectable effect of HIV nef on LTR activity was not the result of mutations in integrated nef DNA, as was shown by polymerase chain reaction. These data suggest that the role of nef in HIV genome transcription does not necessarily involve a direct influence on HIV LTR activity.

Astrocytoma↗
PubMed1997

The octamer-binding proteins Oct-1 and Oct-2 repress the HIV long terminal repeat promoter and its transactivation by Tat.

Although the HIV-1 long terminal repeat (LTR) contains four potential binding sites for the octamer-binding protein, Oct-1, which is known to interact with the HIV-1 Tat protein, the effect of the Oct-1 factor on HIV LTR-driven gene expression has not previously been reported. We show here that both Oct-1, and to a lesser extent the related Oct-2 protein, can repress both the basal activity of the HIV-1 LTR and its transactivation by Tat. These effects are still observed with an HIV LTR construct containing only a single octamer-binding site located between the TATA box and the transcriptional start site. The stronger inhibitory effect of Oct-1 on both these promoters is dependent upon its C-terminal region which cannot be effectively replaced by the equivalent region of Oct-2. These effects are discussed in terms of the regulation of HIV LTR activity in different cell types and in response to T-cell activation.

DNA-Binding Proteins↗
PubMed1992

Antagonistic effect of interferon-gamma on tat-induced transactivation of HIV long terminal repeat.

Interferon-gamma (IFN-gamma) has been shown to inhibit human immunodeficiency virus (HIV) replication in macrophages. However, the site of its effect on the HIV infectious cycle is unknown. We show here that IFN-gamma inhibits the transactivation of HIV long terminal repeat (LTR) during viral infection and that it antagonizes tat effect in HT4LacZ-1 cells. HT4LacZ-1 is an indicator CD4+ HeLa cell line for HIV infectivity, because it harbors a HIV LTR-LacZ gene susceptible to transactivation by tat. It was used in combination with a computer-assisted image analyzer to quantify: (i) the number of transactivated foci following HIV infection, (ii) their individual level of transactivation, and (iii) the fusion potency of infected cells. IFN-gamma induced a 75% decrease of the number of transactivated foci following infection of HT4LacZ-1 cells by HIV. The remaining 25% foci still susceptible to transactivation were transactivated at a lower level than in control cultures, and the fusion potency of infected cells was strongly decreased. IFN-gamma acted after HIV entry into the cell and independently of reverse transcription. IFN-gamma antagonized tat-induced LTR transactivation: it inhibited transactivation of HT4LacZ-1 cells when tat was provided either from a SV40-based expression vector of tat or by polyethylene glycol-induced cell fusion with HeLa-tat-III cells. These results suggest that IFN-gamma affects the expression or the activity of cellular factors interacting with tat and that the high level of IFN-gamma production associated with HIV infection plays a role in the establishment of HIV latency.

Genes, tat↗
PubMed1997

TAR and Sp1-independent transactivation of HIV long terminal repeat by the Tat protein in the presence of human cytomegalovirus IE1/IE2.

OBJECTIVE: The HIV Tat protein is a transcriptional transactivator of the HIV-1 long terminal repeat (LTR) promoter element. Its activity depends on its direct interaction with the trans-activation response (TAR) element, although TAR-independent activation by Tat has been demonstrated in different cells. Herpesviruses in general and human cytomegalovirus (HCMV) in particular are often isolated from HIV-1-infected patients and could play a role in the activation of latent HIV and in a subsequent increase in HIV replication. HCMV immediate early gene products (IE1 and IE2) are nuclear phosphoproteins that play a pivotal role in HCMV replication and have been shown to transregulate both viral and cellular gene expression. It has repeatedly been shown that HCMV IE1/IE2 can independently transactivate HIV-1 LTR. The aim of this study was to investigate IE1/IE2 transactivation of HIV-1 LTR in a CD4+ T-cell line in the absence and presence of HIV-1 Tat to establish whether IE1/IE2 can synergize with Tat. METHODS: HIV-1 LTR transactivation by HCMV IE1/IE2 in the presence and absence of HIV-1 Tat was determined by transient transfection experiments of J-Jhan lymphoblastoid cells with a series of different expression vectors. RESULTS: We found a strong synergistic transactivation between HIV Tat and the IE1-IE2 complex on HIV LTR activity using vectors driven either by wild-type LTR or by the nuclear factor NF-kappa(B) response element-mutated HIV LTR. IE1/IE2 synergism with HIV Tat was also observed in Sp1 binding site-mutated for TAR-deleted LTR, which cannot be activated by Tat alone. This cooperation is abolished when the region in IE2 that binds the TATA box binding protein is deleted. CONCLUSIONS: The results obtained indicate that Sp1-binding and TAR sequences are not strictly required for Tat responsiveness when Tat is directed to the HIV promoter by HCMV IE1-IE2. This synergistic effect is mediated by the IE2 and TATA-binding region, and could play a major role in HIV activation when cells are infected by both viruses, a feature often observed in AIDS patients.

Cytomegalovirus↗
PubMed1996

Selective effects of DNA damaging agents on HIV long terminal repeat activation and virus replication in vitro.

Much attention has recently focused on the observation that UV light can activate the long terminal repeat (LTR) of the human immunodeficiency virus (HIV). Although the mechanism of LTR activation remains obscure, several lines of investigation have suggested that it is a result of activation of the NF-kappa B transcription factor(s) following signaling events related to generalized DNA damage. In this report, we present data demonstrating that HIV LTR activation is not a general consequence of cellular DNA damage, but rather a process unique to specific genotoxic stimuli, and that it does not necessarily depend on activation of NF-kappa B. Furthermore, we demonstrate that several of these agents can significantly increase HIV replication and accelerate CD4-positive lymphocyte cytotoxicity in vitro. These findings, therefore, could have clinical significance to AIDS patients with malignancies who are undergoing radiotherapy and chemotherapy.

Antineoplastic Agents↗
PubMed1996

Activation of the HIV long terminal repeat and viral production by H2O2-vanadate.

The long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) contains sequences required for the initiation of gene transcription. Among the substances known to activate the HIV-1 LTR is hydrogen peroxide (H2O2). We report here that H2O2-induced activation of the LTR in the macrophage cell line THP-1 and the lymphocyte cell line, Jurkat, is greatly increased by vanadate. Activation of the LTR by phorbol myristate acetate, tumor necrosis factor alpha, lipopolysaccharide, or Staphylococcus epidermidis extract was not increased by vanadate, indicating some selectivity for H2O2. H2O2 and vanadate also acted synergistically to increase the production of HIV-1 virions by the latently infected macrophage cell line U-1 as determined by p24 antigen release and the detection of intact virions by electron microscopy. Effects were observed at H2O2 and vanadate concentrations down to 3 x 10(-6) M, with high concentrations leading to cell toxicity. Catalase was strongly inhibitory when added prior to the interaction of H2O2 and vanadate, but was considerably less inhibitory when the H2O2 and vanadate were allowed to preincubate prior to the catalase addition. H2O2 reacts with vanadate to form peroxides of vanadate that have potent biological effects. Our findings suggest that among these is the activation of the HIV-1 LTR.

Catalase↗
PubMed1995

Retinoic acid inhibits both the basal activity and phorbol ester-mediated activation of the HIV long terminal repeat promoter.

OBJECTIVE: To establish whether the steroid hormone retinoic acid (RA) was able to modulate the activity of the HIV-1 promoter. DESIGN: The effect of RA and nuclear factor (NF)-kappa B on HIV-1 promoter activity was investigated using HIV-1 long terminal repeat (LTR)-based reporter constructs. METHODS: The activity of wild type and mutant LTR sequences was compared in a variety of stably and transiently transfected human cell lines. RESULTS: It was shown that RA treatment inhibits both the basal activity of the HIV-1 LTR and its stimulation by phorbol ester treatment. This inhibition is observed in both HeLa cells (in the presence of exogenously supplied RA receptors) and the naturally RA-responsive U937 monocyte cell line. RA can also inhibit the stimulation of the HIV-1 LTR by overexpression of NF-kappa B subunits, while linkage of the NF-kappa B sites in the HIV promoter to a heterologous promoter results in its inhibition by RA. CONCLUSIONS: The data presented clearly demonstrate a negative effect of RA on HIV-1 LTR activity that may contribute to its effect on viral replication.

Base Sequence↗
PubMed1992

Expression directed from HIV long terminal repeats in the central nervous system of transgenic mice.

Infection with the human immunodeficiency virus (HIV) is frequently accompanied by the AIDS (acquired immunodeficiency syndrome) dementia complex. The role of specific HIV genetic elements in the pathogenesis of central nervous system (CNS) disease is not clear. Transgenic mice were constructed that contained the long terminal repeats (LTRs) of two CNS-derived strains and a T cell tropic strain of HIV-1. Only mice generated with CNS-derived LTRs directed expression in the CNS, particularly in neurons. Thus, some strains of HIV-1 have a selective advantage for gene expression in the brain, and neurons can supply the cellular factors necessary for their transcription.

Animals↗
PubMed1987

Transcription directed by the HIV long terminal repeat in vitro.

The long terminal repeat (LTR) of the AIDS virus (HIV) has been found to contain promoter sequences that are active in uninfected HeLa whole cell and nuclear extracts. Here we report that elements upstream of position -104 (start site +1) do not affect transcriptional activity in vitro whereas sequences between -104 and -57 are required for such activity. Using a reconstituted RNA polymerase II system, we demonstrate that a partially purified fraction containing Spl not only stimulates, as was previously reported, but is required for accurate initiation of transcription directed by the HIV LTR. In addition, based on a computerized analysis, we report the presence of a region in the HIV LTR (positions -151 to -80) that is similar to the 72 base pair enhancer element of SV40 and that includes a highly conserved segment also present in the cytomegalovirus enhancer. Moreover, the HIV and HTLV-I LTRs are shown to share a region of similarity that includes the 21 base pair motif found in the enhancers of the human and bovine T-lymphotropic viruses. The R region of the HIV LTR is found to have two extensive regions of dyad symmetry rather than one as was previously reported. The significance of these observations for HIV pathogenesis is discussed.

DNA Restriction Enzymes↗
PubMed1992

Redox status of cells influences constitutive or induced NF-kappa B translocation and HIV long terminal repeat activity in human T and monocytic cell lines.

We have tested the hypothesis that cellular activation events occurring in T lymphocytes and monocytes and mediated through translocation of the transcription factor NF-kappa B are dependent upon the constitutive redox status of these cells. We used phenolic, lipid-soluble, chain-breaking antioxidants (butylated hydroxyanisole (BHA), nordihydroquairetic acid, or alpha-tocopherol (vitamin E) to show that peroxyl radical scavenging in unstimulated and PMA- or TNF-stimulated cells blocks the functions depending on NF-kappa B activation. BHA was found to suppress not only PMA- or TNF-induced, but also constitutive, HIV-enhancer activity concomitant to an inhibition of NF-kappa B binding activity in both lymphoblastoid T (J.Jhan) and monocytic (U937) cell lines. This was also true for KBF (p50 homodimer) binding activity in U937 cells. Secretion of TNF, the product of another NF-kappa B-dependent gene, was abolished by BHA in PMA-stimulated U937 cells. The anti-oxidative effect of BHA was accompanied by an increase in thiol, but not glutathione, content in stimulated and unstimulated T cell, whereas TNF stimulation itself barely modified the cellular thiol level. Oxidative stress obtained by the addition of H2O2 to the culture medium of J.Jhan or U937 cells could not by itself induce NF-kappa B activation. These observations suggest that TNF and PMA do not lead to NF-kappa B activation through induction of changes in the cell redox status. Rather, TNF and PMA can exert their effect only if cells are in an appropriate redox status, because prior modification toward reduction with BHA treatment prevents this activation. It appears that a basal redox equilibrium tending toward oxidation is a prerequisite for full activation of transduction pathways regulating the activity of NF-kappa B-dependent genes.

Base Sequence↗
PubMed1995

Interactions of thyroid hormone receptor with the human immunodeficiency virus type 1 (HIV-1) long terminal repeat and the HIV-1 Tat transactivator.

Thyroid hormone (T3) receptor (T3R) regulates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by binding to and activating thyroid hormone response elements (TREs) embedded within the viral NF-kappa B and Sp1 motifs. The TREs within the NF-kappa B sites are necessary for activation by T3 in the absence of Tat, while those in the Sp1 motifs function as TREs only when Tat is expressed, suggesting that Tat and T3R interact in the cell. Transactivation of the HIV-1 LTR by T3R alpha and several receptor mutants revealed that the 50-amino-acid N-terminal A/B region of T3R alpha, known to interact with the basal transcription factor TFIIB, is critical for activation of both Tat-dependent and Tat-independent responsive sequences of the LTR. A single amino acid change in the highly conserved tau 1 region in the ligand-binding domain of T3R alpha eliminates Tat-independent but not Tat-dependent activation of the HIV-1 LTR by T3. Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one], which inhibits Tat-mediated transactivation of HIV-1, also inhibits the functional interaction between Tat and T3R alpha. Binding studies with glutathione-S-transferase fusion proteins and Western (immunoblot) analysis indicate that T3R alpha interacts with Tat through amino acids within the DNA-binding domain of T3R alpha. Mutational analysis revealed that amino acid residues in the basic and C-terminal regions of Tat are required for the binding of Tat to T3R alpha, while the N terminus of Tat is not required. These studies provide functional and physical evidence that stimulation of the HIV-1 LTR by T3 involves an interaction between T3R alpha and Tat. Our results also suggest a model in which multiple domains of T3R alpha interact with Tat and other factors to form transcriptionally important complexes.

Antiviral Agents↗
PubMed1994

TAT-mediated transcellular activation of HIV-1 long terminal repeat directed gene expression by HIV-1-infected peripheral blood mononuclear cells.

We have previously shown evidence of Tat-mediated transcellular activation of the HIV-1 long terminal repeat (LTR) in vitro by using a laboratory-adapted strain of HIV-1. To examine the biologic significance of this observation, we asked whether primary PBMCs from HIV-1-infected individuals will transactivate the HIV-1 LTR transcellularly in suitable indicator cells. In cultures of PBMCs isolated from HIV-1-infected patients at various clinical stages, with either HIV-1 LTR-transfected Jurkat T cells or nonfusigenic HIV-1 LTR-transfected murine fibroblasts, transcellular activation was readily detected. Transactivation of the LTR in cocultured cells with HIV-1-infected PBMCs is detectable 1 to 2 wk before the onset of significant virus production and at ratios as low as 1 infected cell to 10(6) surrounding cells. Addition of the Tat inhibitor RO5-3335 substantially decreases transcellular activation, even at low concentrations (0.01 microM) that do not affect virus levels. In contrast, addition of the antiretroviral agent zidovudine has no effect on transcellular activation. These data suggest that Tat-mediated transcellular activation of the HIV-1 LTR occurs independently of cellular infection, and provides a mechanism that can promote the spread of HIV-1 in susceptible cell populations.

Cells, Cultured↗
PubMed1991

High expression of exogenous cDNAs directed by HIV-1 long terminal repeat in human cells constitutively producing HIV-1 tat and adenovirus E1A/E1B.

A eukaryotic vector-host cell system is described where the additive transactivating effects of HIV-1 tat and adenovirus E1A on HIV-1 long terminal repeat (LTR) are exploited to increase expression of exogenous cDNAs. Human 143B and 293 cells, the latter constitutively producing E1A, were used as host cell lines. The bacterial gene chloramphenicol acetyltransferase (CAT) and the hepatitis B surface antigen (HBs-Ag) gene were employed as reporter genes inserted in pRPneoU3R, an episomal vector containing BK virus replication origin and early region, where cDNAs are expressed under control of HIV-1 LTR. The 293 cells were transformed by tat expression vectors to constitutively express tat. Stable cell clones of 293tat cells, constitutively expressing CAT after transformation with pRPneoU3R-CAT, show a CAT activity 600-fold higher than normal 293 transformed cells. CAT expression obtained in normal 293 cells can be transiently increased 10-fold by transfection by vectors expressing tat. The 293tat cells transformed by pRPneoU3R-HBs, an episomal vector expressing HBs-Ag from HIV LTR, yielded stable cell clones secreting HBs-Ag in the culture medium at a concentration up to 744 ng/ml or 44 ng/10(6) cells/24 h, 48-fold more than normal 293 cells. The use of this system for constitutive or inducible expression of sequences under control of HIV-1 LTR is discussed in view of possible applications for diagnostic, vaccinal and therapeutic purposes.

Adenovirus Early Proteins↗
PubMed1992

Regulatory elements in the human immunodeficiency virus type 1 long terminal repeat LTR (HIV-1) responsive to steroid hormone stimulation.

Within the long terminal repeat (LTR) of the human immunodeficiency virus type 1 (HIV-1) provirus there exists a steroid hormone-responsive element corresponding to the TGTTCT sequence identified as the glucocorticoid receptor binding element within the LTR of mouse mammary tumor virus. We have used an LTR(HIV-1)-chloramphenicol acetyl transferase (CAT) plasmid construct to transfect infected H9V3 and noninfected H9 cells. Four hours before harvest the cells were divided into two parts and half was treated with hydrocortisone (10(-7) M). The cells were harvested and washed, and the CAT activity was measured. In eight repeat experiments an increased expression of the CAT gene has consistently been observed in H9V3 cells in response to the glucocorticoid but no significant effect of the steroid was observed in noninfected cells. Double transfection of LTR(HIV-1)-TAT and LTR(HIV-1)-CAT into noninfected H9 cells results in a cell population in which the CAT gene was responsive to glucocorticoid stimulation. A time course and dose response for the steroid effect have been determined and the binding of steroid receptor fo the LTR-DNA characterized by gel retardation experiments.

Base Sequence↗
PubMed1989

Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.

The human immunodeficiency virus (HIV-1)-encoded trans-activator (tat) increases HIV gene expression and replication. Previously, we demonstrated that tat facilitates elongation of transcription through the HIV-1 long terminal repeat (LTR) and that short transcripts corresponding to prematurely terminated RNA are released and accumulate in the absence of tat. Here, using a transient expression assay, we tested clustered and compensatory mutations, as well as 3' deletions, in the trans-acting responsive region (tar) and observed that the primary sequence in the loop and secondary structure in the stem of the stem-loop in tar are required for trans-activation by tat. Insertions in the 5' region of tar revealed that tar must be near the site of HIV-1 initiation of transcription for trans-activation by tat. Deletions (3') and an insertion in tar demonstrated that an intact stem-loop is required for the recovery of prematurely terminated transcripts. Short and full-length transcripts were observed also with HIV type 2 (HIV-2) in the absence and presence of tat, respectively. We conclude that an intact stem-loop in tar is essential for trans-activation by tat and that initiation of transcription by HIV-1 promoter factors and elongation of transcription by tat are coupled.

Chloramphenicol O-Acetyltransferase↗
PubMed1990

Effect of tamoxifen on regulation of viral replication and human immunodeficiency virus (HIV) long terminal repeat-directed transcription in cells chronically infected with HIV-1.

The protein kinase C (PKC) activator phorbol myristate acetate (PMA) was used to upregulate viral replication in a clone of promonocytic cells chronically infected with human immunodeficiency virus (HIV)-1. Induction of virus could be inhibited by the triphenylethylene anti-estrogen tamoxifen at concentrations that had minimal effects on cellular DNA synthetic responses and cell cycle kinetics. This effect correlated with tamoxifen's ability to block PMA-mediated enhancement of HIV-promoter-driven transactivation in cells of monocyte and CD4+ T-lymphocyte lineages. No interference with a primary infection was noted. Tamoxifen's mechanism of action may relate both to its capacity to inhibit PKC and to consensus sequences for gonadal steroid responsive elements in the HIV long terminal repeat, as it was able to partially inhibit another HIV activator, 5-azacytidine, which does not modulate PKC function. The finding that regulation of HIV in a model for low-level chronic or latent infection is amenable to a nonimmunosuppressive steroid antagonist may suggest approaches to pharmacologic intervention early in HIV infection.

Cell Cycle↗
PubMed1994

Different members of the Sp1 multigene family exert opposite transcriptional regulation of the long terminal repeat of HIV-1.

Recently, a family of transcription factors structurally related to Sp1 has been described; thus, more than one activator may bind to the GC boxes present in a number of viral and cellular promoters. We have compared the transactivation potentials of Sp1, Sp3 and Sp4 proteins on the human immunodeficiency virus type 1 (HIV-1) promoter. The long terminal repeat (LTR) of HIV-1 contains three binding sites for the transcription factor Sp1 (GC boxes) which are involved in both basal and Tat-mediated transcriptional activation. Moreover, a cooperative interaction between NF-kappa B and Sp1 is required for HIV enhancer activation. We now demonstrate that Sp4 is an activator, while the Sp3 protein represses basal expression of HIV promoter. Remarkably, we found that over-expression of the transcription factor Sp3 was able to suppress Tat-mediated transactivation. These inhibitory effects of Sp3 correlate with its DNA binding activity, suggesting that Sp3 inhibition involves competition with Sp1 for occupancy of the GC boxes. Next, we have analyzed the role of different Sp1-related proteins in the stimulation of HIV-1 promoter in response to mitogens. We found that the binding of NF-kappa B is not by itself sufficient to induce HIV gene expression. Instead, an interaction between NF-kappa B and the trans-acting domain (A domain) of Sp1 bound to an adjacent site must occur. We found that the cooperative interaction between NF-kappa B and Sp1 is highly specific, since neither Sp3 nor Sp4 is capable of cooperating with NF-kappa B.

Base Sequence↗