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Results for “HIV Long Terminal Repeat”

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At least 307 records · Page 17Linked to original sources

PubMed1999

Activation of human immunodeficiency virus type 1 expression by Gardnerella vaginalis.

Bacterial vaginosis (BV) is associated with an increased rate of sexual transmission of human immunodeficiency virus (HIV) type 1, and Gardnerella vaginalis is frequently isolated from the genital tracts of women with BV. G. vaginalis lysates were found to significantly stimulate HIV expression in monocytoid cells. Stimulation was significantly higher when lysates were heated at 100 degrees C for 5 min but was reduced by treatment with lysozyme or protease. G. vaginalis lysates also activated HIV expression in certain T cell lines. G. vaginalis lysates activated HIV long-terminal repeat transcription in HIV-infected cells and increased NF-kappaB binding activity, indicating an effect by G. vaginalis on HIV transcription. The activation of HIV production by G. vaginalis suggests that genital tract infection with G. vaginalis increases the risk of HIV transmission by increasing HIV expression in the genital tract. This may explain, at least in part, the increased rate of HIV transmission in women with BV.

Cytokines↗

PubMed1991

Circular DNA of human immunodeficiency virus: analysis of circle junction nucleotide sequences.

During infection of cells by retroviruses, some of the nonintegrated viral DNA can be found as a circular form containing two tandem, directly repeated long terminal repeats. The nucleotide sequence at the point where the long terminal repeats join (the circle junction) can be used to deduce the terminal nucleotides of the linear form of the viral DNA. Comparison of the termini of linear viral DNA with sequences at the junctions between the integrated provirus and the host chromosome has revealed that for most retroviruses 2 bp are removed from each end of the linear viral DNA during integration. For human immunodeficiency virus type 1 (HIV-1), however, sequence considerations involving primer-binding sites had suggested that only 1 bp is removed during integration. We obtained the nucleotide sequences at the ends of HIV-1 DNA by using the polymerase chain reaction to amplify fragments corresponding to the HIV-1 circle junction. Of 17 clones containing amplified sequences, 10 had identical circle junctions that contained an additional 4 bp (GTAC) relative to the integrated provirus. This indicates that, as for other retroviruses, 2 bp are removed from each end of the linear HIV-1 viral DNA during integration. The remaining seven isolates contained insertions or deletions at the circle junction.

Base Sequence↗

PubMed2002

HIV Tat binds Egr proteins and enhances Egr-dependent transactivation of the Fas ligand promoter.

HIV Tat can enhance activation-induced up-regulation of Fas ligand (FasL), which may contribute to T cell apoptosis in human immune deficiency virus (HIV)-infected individuals. We have assessed functional and physical interactions between Tat and the Egr family of transcription factors (Egr-1, -2, and -3), the latter two of which are major participants in activation-induced FasL up-regulation. Here we report that whereas Tat itself has no effect on the FasL promoter, it binds to Egr-2 and -3 and synergizes with them to superinduce expression of a FasL promoter-driven reporter. A Tat molecule containing a single amino acid substitution that results in the loss of transactivation activity for the HIV long terminal repeat still binds Egr-3 but can no longer enhance Egr-mediated transactivation of the FasL promoter. Furthermore, the mutated Tat acts as a dominant negative inhibitor, blocking the superinduction of FasL caused by wild type Tat. Because Tat is present in virus-infected cells and in the serum of HIV-infected individuals, these results suggest that increased expression of FasL in these circumstances may result from the cooperative activities of activation-induced Egrs and Tat.

DNA, Complementary↗

PubMed2003

Transcriptional profiles of latent human immunodeficiency virus in infected individuals: effects of Tat on the host and reservoir.

The persistence of human immunodeficiency virus (HIV) in optimally treated infected individuals poses a major therapeutic problem. In latently infected cells, one of the observed phenotypes is absent elongation of viral transcription. Thus, the positive elongation factor b (P-TEFb), which is usually recruited by NF-kappaB or Tat, is not present on the HIV long terminal repeat (LTR). Although most attempts to activate these proviruses centered on NF-kappaB, we investigated effects of Tat. To this end, we generated transgenic mice, which secreted a chimera between Tat and the green fluorescent protein from beta cells of the pancreas. This extracellular Tat distributed widely, entered nuclei of resting cells, and specifically transactivated the HIV LTR. No deleterious side effects of Tat were found. Next, we determined that Tat can activate latent proviruses in optimally treated infected individuals. In their cells, T-cell activation or exogenous Tat could induce viral replication equivalently. Thus, P-TEFb could activate the majority of the latent HIV, in this case by Tat.

Animals↗

PubMed1995

Molecular and functional interactions of transcription factor USF with the long terminal repeat of human immunodeficiency virus type 1.

The human transcription factor USF, purified from HeLa cells, and its recombinant 43-kDa component bind to the long terminal repeat (LTR) of human immunodeficiency virus type 1. The proteins footprint over nucleotides from position -173 to -157 upstream of the transcription start site, generating strong DNAse I hypersensitivity sites at the 3' sides on both strands. As detected by methylation protection studies, the factor forms symmetric contacts with the guanines of the palindromic CACGTG core of the recognized sequence. Its binding ability is abolished by the mutation of this core sequence and is strongly reduced by the cytosine methylation of the central CpG dinucleotide. Upon binding, both recombinant and purified USFs bend the LTR DNA template. The role of USF in the control of transcription initiation from the LTR was tested by in vitro transcription assays. Upon addition of the protein, transcription from constructs containing an intact binding site is increased, while the responsiveness in constructs with a mutated sequence is abolished. Furthermore, the addition of a decoy plasmid which contains multiple repeats of the target sequence results in downregulation of transcription from the LTR. These results suggest that USF is a positive regulator of LTR-mediated transcriptional activation.

Base Sequence↗

PubMed1997

Transmission of HIV-1 in infants born to seropositive mothers: PCR-amplified proviral DNA detected by flow cytometric analysis of immunoreactive beads.

The diagnosis of HIV infection in newborns is established by amplification of proviral DNA using the polymerase chain reaction (PCR). We developed a nonisotopic method for heminested PCR using a biotinylated primer among sets of three oligonucleotides, each selected from the HIV long terminal repeat (LTR) and gag sequences. An internal probe incorporating digoxigenin-dUTP was also synthesized by PCR. The PCR products, hybridized with LTR region or gag region probes, were captured with streptavidin-coated magnetic beads and detected by fluorescein isothiocyanate-labeled antidigoxigenin in flow cytometric analysis. This immunoreactive bead assay (PCR-IRB) detected about three copies of HIV proviral DNA. A panel of 50 coded DNA specimens of infants previously assayed by conventional PCR and with known clinical results revealed that the PCR-IRB findings using LTR, but not gag, were in agreement. A double-blind prospective study of blood samples from 14 mother-infant pairs using the PCR-IRB amplification of LTR gave results similar to the commercial Amplicor HIV-1 PCR test and were consistent with the clinical outcomes. PCR-IRB results were positive for 11 mothers and three infants, one at birth, one at 2 weeks after birth, and one at 8 weeks after birth. PCR-IRB is a simple, reliable, specific, and automatable assay useful in the early diagnosis of perinatal HIV infection in clinical practice and regional screening programs.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene.

The cDNAs for two DNA binding proteins of BTE, a GC box sequence in the promoter region of the P-450IA1(CYP1A1) gene, have been isolated from a rat liver cDNA library by using the BTE sequence as a binding probe. While one is for the rat equivalent to human Sp1, the other encodes a primary structure of 244 amino acids, a novel DNA binding protein designated BTEB. Both proteins contain a zinc finger domain of Cys-Cys/His-His motif that is repeated three times with sequence similarity of 72% to each other, otherwise they share little or no similarity. The function of BTEB was analysed by transfection of plasmids expressing BTEB and/or Sp1 with appropriate reporter plasmids into a monkey cell line CV-1 and compared with Sp1. BTEB and Sp1 activated the expression of genes with repeated GC box sequences in promoters such as the simian virus 40 early promoter and the human immunodeficiency virus-1 long terminal repeat promoter. In contrast, BTEB repressed the activity of a promoter containing BTE, a single GC box of the CYP1A1 gene that is stimulated by Sp1. When the BTE sequence was repeated five times, however, BTEB turned out to be an activator of the promoter. RNA blot analysis showed that mRNAs for BTEB and Sp1 were expressed in all tissues tested, but their concentrations varied independently in tissues. The former mRNA was rich in the brain, kidney, lung and testis, while the latter was relatively abundant in the thymus and spleen.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

NF-kappa B activates the HIV promoter in neurons.

Human immunodeficiency virus (HIV) infection of the brain leads to massive neuronal damage, resulting in the AIDS (acquired immunodeficiency syndrome) dementia complex (ADC). A recent study using transgenic mice indicates that neurons possess transcription factors capable of activating the HIV promoter. To identify these, we transfected two types of primary cultures of rat neurons with HIV promoter-reporter gene constructs. The two kappa B regulatory sites in the HIV long terminal repeat (LTR) are shown to be essential for strong promoter activity. Two proteins present in neurons, BETA and an NF-kappa B-like protein, can bind the kappa B sites. These proteins are shown to belong to distinct families of transcription factors. Mutation analysis and transfection of a dominant negative NF-kappa B mutant, indicate that the neuronal NF-kappa B-like activity mediates HIV promoter activation. cDNA cloning, biochemical and immunological analyses indicate that neuronal NF-kappa B is similar to NF-kappa B of other tissues. Transfections of primary neuron cultures with an HIV promoter-beta-galactosidase construct show that within these cultures, neurons are indeed the cells that highly activate the HIV promoter. Thus, analogous to the situation in T-lymphocytes and macrophages, NF-kappa B is an activator of HIV transcription in neurons.

AIDS Dementia Complex↗

PubMed2005

Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system.

We describe a phased screening system for discovery of compounds with antiviral activity against hepatitis C virus (HCV). The primary assay utilizes dicistronic subgenomic HCV replicons in which the upstream cistron was modified to express the human immunodeficiency virus (HIV) tat protein. When these replicons are stably transfected into Huh-7-derived cells that express secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV long terminal repeat promoter, there is a strong correlation between intracellular HCV RNA abundance and the activity of SEAP secreted into the culture medium. Thus, active compounds are easily identified by direct enzymatic quantification of SEAP in the medium without post-assay processing. Compounds that reduce SEAP activity without causing cellular toxicity are next evaluated in a second Huh-7-derived cell line constitutively expressing SEAP under control of the tat-HIV promoter axis, independent of HCV RNA replication. This specificity control identifies compounds that cause reductions in SEAP that are unrelated to suppression of HCV RNA replication. Compounds showing HCV-specific activity in primary assays are next evaluated by real-time RT-PCR to directly quantify reductions in HCV RNA. We have found excellent agreement between the SEAP and RT-PCR assays. This phased system provides an efficient and cost-effective screen for compounds with antiviral activity against HCV.

Alkaline Phosphatase↗

PubMed2000

Gene activation by Varicella-zoster virus IE4 protein requires its dimerization and involves both the arginine-rich sequence, the central part, and the carboxyl-terminal cysteine-rich region.

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for those of heterologous viruses. Since most transcription factors act as dimers, IE4 dimerization was studied using the mammalian two-hybrid system. Introduction of mutations in the IE4 open reading frame demonstrated that both the central region and the carboxyl-terminal cysteine-rich domain were important for efficient dimerization. Within the carboxyl-terminal domain, substitution of amino acids encompassing residues 443-447 totally abolished dimerization. Gene activation by IE4 was studied by transient transfection with an IE4 expression plasmid and a reporter gene under the control of either the human immunodeficiency virus, type 1, long terminal repeat or the VZV thymidine kinase promoter. Regions of IE4 important for dimerization were also shown to be crucial for transactivation. In addition, the arginine-rich domains Rb and Rc of the amino-terminal region were also demonstrated to be important for transactivation, whereas the Ra domain as well as an acidic and bZIP-containing regions were shown to be dispensable for gene transactivation. A nucleocytoplasmic shuttling of IE4 has also been characterized, involving a nuclear localization signal identified within the Rb domain and a nuclear export mechanism partially depending on Crm-1.

Amino Acid Sequence↗

PubMed1993

Human immunodeficiency virus type 1 transactivator protein, tat, stimulates transcriptional read-through of distal terminator sequences in vitro.

The human immunodeficiency virus type 1 transactivator protein, tat, specifically stimulates transcription from the viral long terminal repeat. We used cell-free transcription systems to test whether tat can stimulate transcriptional read-through of an artificial terminator sequence (e.g., a stable RNA stem-loop structure followed by a tract of nine uridine residues) placed downstream of the viral long terminal repeat. In the absence of tat, RNA polymerases are prematurely released from the template at the terminator sequence. Recombinant tat protein purified from Escherichia coli increased the synthesis of full-length transcripts approximately 25-fold and decreased the amount of transcripts ending at the terminator sequence. The reaction is strictly dependent upon the presence of a functional transactivation-responsive region (TAR) sequence. Mutations in the tat binding site on TAR RNA and mutations in the TAR RNA loop block transactivation in vivo. Neither type of mutation is able to respond to tat in vitro. These results strongly suggest that after transcription through the TAR region, tat modifies the transcription complex to increase its elongation capacity.

Base Sequence↗

PubMed1993

In vivo footprinting analysis of constitutive and inducible protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1.

The regulation of the rate of transcription of human immunodeficiency virus type 1 is mainly exerted through the long terminal repeat (LTR) at the 5' end of the provirus. A large number of cis-acting regulatory elements have been identified in the LTR by in vitro binding studies; the biological role of these sites within living infected cells, however, is still not clear. We have studied the interactions of nuclear proteins with the LTR in the U1 monocytic cell line by in vivo dimethylsulfate footprinting, using the ligation-mediated polymerase chain reaction technique. In this cell line, transcription of the virus, which is very low under basal conditions, is highly inducible by treatment with phorbol esters; therefore, this system is likely to represent a suitable cellular model to study viral latency. Independently of the level of viral transcription, major in vivo footprints appear at the two Sp1 sites adjacent to the enhancer, the downstream-positioned enhancer repeat, the NFAT binding site, and one of the purine-rich sites of the negative regulatory element. Upon transcriptional activation by phorbol myristate acetate, the only perturbation in the footprinting pattern is a dramatic increase in dimethylsulfate sensitivity of guanine at position -92 in the downstream enhancer repeat. This modification is correlated with the transient induction of two enhancer-binding activities, as determined by gel retardation assays. While the transcriptional rate is still increasing and the in vivo footprinting pattern is unchanged at up to 24 h postactivation, these enhancer-binding factors are considerably reduced at this time. Therefore, further levels of regulation have to be considered to explain the maintenance of the induced state.

Base Sequence↗

PubMed1989

Human immunodeficiency virus tat-activated expression of poliovirus protein 2A inhibits mRNA translation.

To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was cotransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

Chloramphenicol O-Acetyltransferase↗

PubMed2005

Identification of attenuated variants of HIV-1 circulating recombinant form 01_AE that are associated with slow disease progression due to gross genetic alterations in the nef/long terminal repeat sequences.

We identified an unusual case of human immunodeficiency virus type 1 (HIV-1) infection in a patient (GM43) who exhibited a persistently low antibody response and undetectable viral load during a 5-year follow-up period. GM43 harbored HIV-1 circulating recombinant form 01_AE with gross deletions in the nef/long terminal repeat (LTR) region. The sizes of the deletions increased progressively from 84 to >400 bp during the 5-year period. GM43 appeared to have acquired defective variants from her husband. The genetic alterations in the nef/LTR region were remarkably similar to those that have been reported in slow progressors (such as the slow progressors in the Sydney Blood Bank Cohort). The present study is the first report of slow disease progression due to gross genetic alterations in the nef/LTR region in a person infected with an HIV-1 non-subtype B strain.

Adult↗

PubMed1994

Flow cytometric immunodetection of human immunodeficiency virus type 1 proviral DNA by heminested PCR and digoxigenin-labeled probes.

PCR is the most sensitive and direct method for detecting blood-borne viruses, as well as an efficient means for producing vector-free probes. However, the application of PCR, especially in the laboratory diagnosis of human immunodeficiency virus (HIV) infection, is impeded by the current use of radiolabeled oligonucleotide probes. Therefore, we have developed a nonisotopic PCR immunoreactive bead (PCR-IRB) assay to detect HIV type 1 proviral DNA from peripheral blood mononuclear cells (PBMC). We used a biotinylated primer in a set of three oligonucleotides selected from the HIV long terminal repeat region for heminested PCR amplification. An internal probe was synthesized by PCR with incorporation of digoxigenin-labeled dUTP. After solution hybridization of the probe with PCR-amplified products (amplicons), the hybridized DNA was captured with streptavidin-coated magnetic beads. For the detection of hybrids, flow cytometric analyses were carried out by two procedures: (i) direct detection with fluorescein isothiocyanate (FITC)-labeled antidigoxigenin immunoglobulin G (IgG) antibody and (ii) indirect detection with antidigoxigenin sheep IgG antibody followed by FITC-labeled anti-sheep IgG antibody. Both procedures in the PCR-IRB assay detected two to three copies of HIV proviral DNA sequences, a sensitivity that is comparable with that of the conventional radioactive detection of amplicons following probe hybridization and electrophoresis. To compare the PCR-IRB assay with the conventional method, we tested 53 pedigreed PBMC specimens from blood donors and newborns; the results obtained were identical. This nonisotopic PCR-IRB assay can also be automated for potential application in laboratory diagnosis of HIV infection, blood bank screening, and therapeutic monitoring of viremia and perinatal transmission.

Base Sequence↗

PubMed1990

Cell-specific differences in activation of NF-kappa B regulatory elements of human immunodeficiency virus and beta interferon promoters by tumor necrosis factor.

Three aspects of the involvement of tumor necrosis factor in human immunodeficiency virus (HIV) pathogenesis were examined. Tumor necrosis factor alpha (TNF-alpha) mRNA production was analyzed by polymerase chain reaction amplification in monocytic U937 cells and in a chronically HIV infected U937 cell line (U9-IIIB). TNF-alpha RNA was undetectable in U937 cells, whereas a low constitutive level was detected in U9-IIIB cells. Paramyxovirus infection induced a 5- to 10-fold increase in the steady-state level of TNF-alpha RNA in U9-IIIB cells compared with U937 cells, suggesting that HIV-infected monocytic cells produced higher levels of TNF-alpha than did normal cells after a secondary virus infection. The effects of TNF-alpha on gene expression were examined by transient expression assays using reporter chloramphenicol acetyltransferase plasmids linked to regulatory elements from the HIV long terminal repeat (LTR) and the beta interferon promoter. In U937 and Jurkat T lymphoid cells, the inducibility of the different hybrid promoters by TNF-alpha or phorbol ester varied in a cell type- and promoter context-specific manner; the levels of gene activity of NF-kappa B-containing plasmids correlated directly with induction of NF-kappa B DNA-binding activity. Although the intact beta interferon promoter was only weakly stimulated by phorbol ester or TNF-alpha, multimers of the PRDII NF-kappa B-binding domain were inducible by both agents. TNF-alpha was able to increase expression of the HIV LTR in T cells, but in monocytic cells, TNF-alpha did not induce the HIV LTR above a constitutive level of activity. This level of NF-kappa B-independent activity appears to be sufficient for virus multiplication, since TNF-alpha treatment had no effect on the kinetics of de novo HIV type 1 (HIV-1) infection and viral RNA production in U937 cells. However, in Jurkat cells, TNF-alpha dramatically enhanced the spread of HIV-1 through the cell population and increased viral RNA synthesis, indicating that in T cells HIV-1 multiplication was stimulated by TNF-alpha treatment.

Base Sequence↗

PubMed1992

A high-level expression vector for human cells.

We have developed a vector (pSupexp), for high-level expression of genes, that is dependent on transactivation of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) by the HIV-1 transactivator protein, Tat. The foreign gene, expressed under transcriptional control of the HIV-1 LTR, and the tat gene, expressed under transcriptional control of SV40 early promoter, are expressed from the same plasmid. The vector also has the neomycin resistance-encoding gene (neo), with G418 being used as a dominant selection marker for stable expression. We have cloned the bacterial cat gene into pSupexp and measured transient CAT production in human HeLa and A549 cells. Our results indicate that pSupexpCAT expresses about 25- to 68-fold higher levels of CAT activity as compared to other standard SV40- and Rous sarcoma virus-based vectors, and three- to fivefold more activity than the cytomegalovirus-based vector. Immunoprecipitation of the CAT protein also revealed a high level of production in human cells.

Cell Line↗

PubMed2001

Modulation of cellular and viral gene expression by the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is the likely etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Common to these malignancies is that tumor cells are latently infected with KSHV. Viral gene expression is limited to a few genes, one of which is the latency-associated nuclear antigen (LANA), the product of ORF73. Examination of the primary sequence of LANA reveals some structural features reminiscent of transcription factors, leading us to hypothesize that LANA may regulate viral and cellular transcription during latency. In reporter gene-based transient transfection assays, we found that LANA can have either positive or negative effects on gene expression. While expression of a reporter gene from several synthetic promoters was increased in the presence of LANA, expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR)-and from NF-kappaB-dependent reporter genes-was reduced by LANA expression. In addition, the promoter of KSHV ORF73 itself is activated up to 5.5-fold by LANA. This autoregulation may be important in tumorigenesis, because two other genes (v-cyclin and v-FLIP) with likely roles in cell growth and survival are also controlled by this element. To identify cellular genes influenced by LANA, we employed cDNA array-based expression profiling. Six known genes (and nine expressed sequence tags) were found to be upregulated in LANA-expressing cell lines. One of these, Staf-50, is known to inhibit expression from the HIV LTR; most of the other known genes are interferon inducible, although the interferon genes themselves were not induced by LANA. These data demonstrate that LANA expression has effects on cellular and viral gene expression. We suggest that, whether direct or indirect in origin, these effects may play important roles in the pathobiology of KSHV infection.

Animals↗