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Results for “HIV Long Terminal Repeat”

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At least 19 recordsLinked to original sources

PubMed2001

NF-kappaB cis-acting motifs of the human immunodeficiency virus (HIV) long terminal repeat regulate HIV transcription in human macrophages.

The role of NF-kappaB in the reactivation of human immunodeficiency virus (HIV) from latency in CD4 T lymphocytes is well documented. However, its role in driving HIV transcription in human macrophages, which contain a constitutive nuclear pool of NF-kappaB, is less well understood. In this study we have investigated the role that the constitutive pool of NF-kappaB and the NF-kappaB cis-acting motifs of the HIV long terminal repeat (LTR) play in regulating HIV transcription in human monocytic cells and primary macrophages. Inhibition of the constitutive nuclear pool of NF-kappaB (RelA and RelB) in the promonocytic U937 cell line using dominant-negative IkappaBalpha significantly decreases HIV replication. Moreover, it is demonstrated that in the differentiated monocytic cell line THP1, which contains a constitutive nuclear pool of NF-kappaB (RelB),an HIV provirus containing mutations of the kappaB cis-acting sites in the LTR is transcriptionally impaired. Reduction of the constitutive pool of NF-kappaB in human macrophages by an adenovirus vector expressing a dominant-negative IkappaBalpha also reduces HIV transcription. Lastly, mutation of the NF-kappaB cis-acting sites in the LTR of an R5 HIV provirus completely abrogates the first cycle of HIV transcription. These studies indicate that the cis-acting NF-kappaB motifs of the HIV LTR are critical in initiating HIV transcription in human macrophages and suggest that the constitutive nuclear pool of NF-kappaB is important in regulating HIV transcription in these cells.

Base Sequence↗

PubMed2003

Toll-like receptor 2 (TLR2) and TLR9 signaling results in HIV-long terminal repeat trans-activation and HIV replication in HIV-1 transgenic mouse spleen cells: implications of simultaneous activation of TLRs on HIV replication.

Opportunistic infections are common in HIV-infected patients; they activate HIV replication and contribute to disease progression. In the present study we examined the role of Toll-like receptor 2 (TLR2) and TLR9 in HIV-long terminal repeat (HIV-LTR) trans-activation and assessed whether TLR4 synergized with TLR2 or TLR9 to induce HIV replication. Soluble Mycobacterium tuberculosis factor (STF) and phenol-soluble modulin from Staphylococcus epidermidis induced HIV-LTR trans-activation in human microvessel endothelial cells cotransfected with TLR2 cDNA. Stimulation of ex vivo spleen cells from HIV-1 transgenic mice with TLR4, TLR2, and TLR9 ligands (LPS, STF, and CpG DNA, respectively) induced p24 Ag production in a dose-dependent manner. Costimulation of HIV-1 transgenic mice spleen cells with LPS and STF or CpG DNA induced TNF-alpha and IFN-gamma production in a synergistic manner and p24 production in an additive fashion. In the THP-1 human monocytic cell line stably expressing the HIV-LTR-luciferase construct, LPS and STF also induced HIV-LTR trans-activation in an additive manner. This is the first time that TLR2 and TLR9 and costimulation of TLRs have been shown to induce HIV replication. Together these results underscore the importance of TLRs in bacterial Ag- and CpG DNA-induced HIV-LTR trans-activation and HIV replication. These observations may be important in understanding the role of the innate immune system and the molecular mechanisms involved in the increased HIV replication and HIV disease progression associated with multiple opportunistic infections.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Rac1 and Toll-IL-1 receptor domain-containing adapter protein mediate Toll-like receptor 4 induction of HIV-long terminal repeat.

Opportunistic infections, common in HIV-1-infected patients, increase HIV replication; however, the intracellular signaling mechanisms involved are not clearly known. We have shown that Toll-like receptor 2 (TLR2), TLR4, and TLR9 mediate microbial Ag-induced HIV-long terminal repeat (HIV-LTR) trans-activation and HIV-1 replication, and that LPS-induced HIV-LTR trans-activation is mediated through myeloid differentiation adapter protein. Recently, Toll-IL-1R domain-containing adapter protein (TIRAP) has been identified as an adapter molecule that mediates responses to TLR2 and TLR4 ligands, and TIRAP was suggested to provide signaling specificity for different TLRs. Rac1, a small GTP-binding protein that is activated upon LPS stimulation of macrophages, activates phosphatidylinositol 3-kinase and Akt and leads to NF-kappaB activation. The roles of Rac1 and TIRAP in LPS activation of HIV replication is not known. In the present study we show that LPS stimulation of human microvessel endothelial cells leads to Rac1 activation. Constitutively active Rac1 (Rac1V12) simulated the effect of LPS to activate HIV-LTR, whereas the expression of dominant negative Rac1 (Rac1N17) partially blocked LPS-induced HIV-LTR trans-activation. Rac1V12-induced HIV-LTR activation was independent of myeloid differentiation adapter protein, and dominant negative TIRAP blocked Rac1V12-induced HIV-LTR trans-activation. In this study we show for the first time that activation of Rac1 leads to HIV-LTR trans-activation, and this is mediated through TIRAP. Together these results underscore the importance of Rac1 and TIRAP in TLR4 activation of HIV replication and help delineate the signaling pathways induced by TLRs to mediate microbial Ag-induced HIV replication and HIV pathogenesis.

Cells, Cultured↗

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↗

PubMed1997

Selective inhibition of mitogen-induced transactivation of the HIV long terminal repeat by carboxyamidotriazole. Calcium influx blockade represses HIV-1 transcriptional activation.

Carboxyamidotriazole (CAI) is a calcium influx inhibitor that has both antiproliferative and antimetastatic activities. Pretreatment of human T-cells with micromolar concentrations of CAI causes a near complete inhibition of calcium-regulated mitogen-induced transcription from the human immunodeficiency virus (HIV) long terminal repeat (LTR). This inhibition is selective since other mitogen-activated gene regulatory elements, such as the 12-O-tetradecanoylphorbol-13-acetate response element, are not influenced by the drug. HIV LTR transcription inhibition is maximal at 1.0 microM CAI, requires a pretreatment interval of at least 8 h for optimum inhibition, and shows no acute interference with the growth properties of the cells. Moreover, the inhibition is rapidly reversible upon removal of the drug from the medium. Studies to identify enhancer elements within the HIV LTR that are functionally sensitive to low-dose long-term pretreatment with CAI indicate that the NF-kappaB-binding sites are among the major targets of drug action. In vitro DNA binding studies with nuclear extracts prepared from mitogen-induced T-cells stimulated in the presence of CAI indicate that the drug differentially influences the calcium-regulated downstream signal transduction pathways necessary for specific NF-kappaB DNA binding activity at the two kappaB sites within the HIV LTR. Studies with ionomycin and thapsigargin show that repression is specific for selected modes of inducible calcium entry and indicate that, in T-cells, a major mechanism of CAI action is to modulate calcium influx at a level that is proximal to the regulated release of calcium from intracellular stores. Measurement of calcium influx in CAI-treated cells reveals a dramatic and reversible inhibition of mitogen-induced calcium influx. These results indicate that CAI can be an important and effective pharmacological tool for analysis of the calcium-dependent modulation of HIV LTR transcription.

Anti-HIV Agents↗

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↗

PubMed2002

The kappa B DNA sequence from the HIV long terminal repeat functions as an allosteric regulator of HIV transcription.

NF-kappaB is an inducible transcription factor involved in the immune response, inflammation, and viral transcription. To address how the two NF-kappaB and three Sp1 binding sites of the human immunodeficiency virus (HIV) long terminal repeat (LTR) control multiple activator assembly and transcription, we first observed and compared unique conformations between the crystallographic structure of the NF-kappaB p50.p65 heterodimer bound to the uPA-kappaB target site to that of the p50.p65.HIV-kappaB complex. Next, cooperativity between two NF-kappaB molecules bound to tandem HIV-kappaB sequences was measured as well as that of NF-kappaB and transcription factor Sp1 when bound to adjacent sites. The cooperativity of hybrid HIV-LTR enhancers was measured with the 3' kappaB site converted to uPA-kappaB or to interferon beta gene enhancer kappaB. The hybrids were defective in transcriptional activator assembly and less active transcriptionally. These functional differences correlate with observed conformational differences and demonstrate that distinct kappaB DNA sequences function as allosteric regulators in a gene-specific manner.

Allosteric Regulation↗

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↗

PubMed2000

Hydroxyurea inhibits the transactivation of the HIV-long-terminal repeat (LTR) promoter.

HIV-1 gene expression is regulated by the promoter/enhancer located within the U3 region of the proviral 5' LTR that contains multiple potential cis-acting regulatory sites. Here we describe that the inhibitor of the cellular ribonucleoside reductase, hydroxyurea (HU), inhibited phorbol myristate acetate- or tumour necrosis factor-alpha-induced HIV-1-LTR transactivation in both lymphoid and non-lymphoid cells in a dose-dependent manner within the first 6 h of treatment, with a 50% inhibitory concentration of 0.5 mM. This inhibition was found to be specific for the HIV-1-LTR since transactivation of either an AP-1-dependent promoter or the CD69 gene promoter was not affected by the presence of HU. Moreover, gel-shift assays in 5.1 cells showed that HU prevented the binding of the NF-kappaB to the kappaB sites located in the HIV-1-LTR region, but it did not affect the binding of both the AP-1 and the Sp-1 transcription factors. By Western blots and cell cycle analyses we detected that HU induced a rapid dephosphorylation of the pRB, the product of the retinoblastoma tumour suppressor gene, and the cell cycle arrest was evident after 24 h of treatment. Thus, HU inhibits HIV-1 promoter activity by a novel pathway that implies an inhibition of the NF-kappaB binding to the LTR promoter. The present study suggests that HU may be useful as a potential therapeutic approach for inhibition of HIV-1 replication through different pathways.

Antigens, CD↗

PubMed2001

Bacterial lipopolysaccharide activates HIV long terminal repeat through Toll-like receptor 4.

In HIV-infected patients, concurrent infections with bacteria and viruses are known to induce HIV replication as assessed by increases in plasma HIV RNA levels. In the present study, we determined the cell surface receptor and molecular mechanisms of enterobacterial LPS-induced HIV transcription. Human dermal microvessel endothelial cells (HMEC) were transfected with an HIV-long terminal repeat (LTR)-luciferase construct and subsequently stimulated with purified bacterial LPS. Our studies demonstrate that human Toll-like receptor 4 (TLR4) mediates LPS-induced NF-kappaB and HIV-LTR activation in HMEC through IL-1 signaling molecules, namely myeloid differentiation protein, IL-1R-associated kinase, TNFR-associated factor, and NF-kappaB-inducing kinase. Cotransfection of HMEC with HIV-LTR-luciferase and TLR4 cDNA from LPS-hyporesponsive C3H/HeJ mice abrogates LPS-induced HIV transcription as does the use of dominant-negative mutants of the IL-1 signaling molecules. Transfection of HMEC with an HIV-LTR-mutant that lacks the NF-kappaB binding site or pretreatment of cells with chemical inhibitors of the NF-kappaB pathway also blocked LPS-induced HIV-LTR transactivation. These data support the conclusion that TLR4 mediates enterobacterial LPS-induced HIV transcription via IL-1 signaling molecules and NF-kappaB activation plays an important role in HIV-LTR transactivation.

Animals↗

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↗