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PubMed1999

High polymorphism level of genomic sequences flanking insertion sites of human endogenous retroviral long terminal repeats.

The polymorphism at the multitude of loci adjacent to human endogenous retrovirus long terminal repeats (LTRs) was analyzed by a technique for whole genome differential display based on the PCR suppression effect that provides selective amplification and display of genomic sequences flanking interspersed repeated elements. This strategy is simple, target-specific, requires a small amount of DNA and provides reproducible and highly informative data. The average frequency of polymorphism observed in the vicinity of the LTR insertion sites was found to be about 12%. The high incidence of polymorphism within the LTR flanks together with the frequent location of LTRs near genes makes the LTR loci a useful source of polymorphic markers for gene mapping.

Chromosome Mapping↗
PubMed1998

Sp1 binding sites inserted into the rous sarcoma virus long terminal repeat enhance LTR-driven gene expression.

Although the Rous sarcoma virus (RSV) long terminal repeat (LTR) is an efficient promoter of transcription, most RSV proviruses are down-regulated upon retroviral integration in non-permissive mammalian cells. Among other mechanisms, DNA methylation has been shown to be involved in proviral silencing. The presence of Sp1 binding sites has been demonstrated to be essential for protection of a CpG island and also non-island DNA regions from de novo methylation. Also, the presence of these sites in the LTRs correlates with the transcriptional activity of certain proviral structures. Using transient and stable transfection assays, we demonstrate that insertion of Sp1 binding sites into the RSV LTR remarkably increases expression of the LTR-driven genes in permissive and non-permissive cells, despite the reported negative effect of insertion of the non-specific DNA into the LTR promoter/enhancer sequences. Particular arrangement of inserted Sp1 sites was effective even in stably transfected reporter gene constructs into non-permissive mammalian cells, where additional factors exert negative effects on expression.

Animals↗
PubMed1989

Human immunodeficiency viral long terminal repeat is functional and can be trans-activated in Escherichia coli.

The long terminal repeat (LTR) of the human immunodeficiency virus (HIV) contains the viral promoter, which is responsible for viral gene expression in eukaryotic cells. We have demonstrated that HIV LTR can also function as a promoter in Escherichia coli. A recombinant plasmid containing the HIV LTR linked to the chloramphenicol acetyltransferase gene can express the enzyme efficiently upon transformation into bacteria. Mung bean nuclease analysis mapped the bacterial transcriptional start site of the promoter to the U3 region of the LTR, in contrast to transcription in eukaryotic cells, which initiates in the U3-R boundary of the LTR. The HIV LTR, besides being fully functional in E. coli, can also be specifically trans-activated by the HIV tat gene product. Trans-activation is demonstrated by an increase in chloramphenicol acetyltransferase activity as well as an increase in the mRNA level of the enzyme. This trans-activation of HIV LTR by tat protein in bacteria offers a useful system to investigate further the specific interaction between tat protein with HIV LTR and the mechanisms of trans-activation.

Base Sequence↗
PubMed1993

Analysis of the DNA topoisomerase-II-mediated cleavage of the long terminal repeat of Drosophila 1731 retrotransposon.

The interaction of DNA topoisomerase II with the long terminal repeat (LTR) of the Drosophila melanogaster 1731 retrotransposon was studied. The covalent binding of topoisomerase II to the LTR was strongly stimulated by different inhibitors of the enzyme 4'-demethylepipodophyllotoxin-9-(4,6-O-2-ethylidene-beta-D-glucopy ranoside (VP-16), 4'-(9-acridinylamino)methanesulfon-m-anisidine) (m-AMSA) and an ellipticine derivative. Enzyme-mediated DNA cleavage could be observed in the absence of inhibitors and was stimulated in their presence. Cleavage occurred predominantly at sites located within or at the boundary of alternating purine/pyrimidine tracts in agreement with previous observations [Spitzner, J. R., Chung, I. K. & Muller, M. T. (1990) Eukaryotic topoisomerase II preferentially cleaves alternating purine-pyrimidine repeats, Nucleic Acids Res. 18, 1-11]. In addition, all of the cleavage sites observed in the absence of inhibitor were located in the U3 region of the LTR. The site specificity of drug-induced cleavage was studied and the conformity of the cleavage sites with previously established consensus sequences was examined. Our results suggest that DNA topoisomerase II, through its ability to alter the degree of DNA supercoiling, might be involved in the control of different functions of the LTR.

Amsacrine↗
PubMed1990

Role of protein kinase A in tax transactivation of the human T-cell leukemia virus type I long terminal repeat.

The human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) is inducible both by the retroviral tax gene product and by cyclic AMP in the murine thymoma S49 cell line. The cis-acting sequences that control transcriptional induction by tax and by cyclic AMP are in close proximity within the HTLV-I promoter. By using a protein kinase A (PKA)-deficient S49 mutant cell line, the response of the viral promoter to cyclic AMP was shown to depend on PKA, whereas the response to tax did not require the activity of this enzyme. Transactivation of the HTLV-I LTR by tax, however, decreased in PKA-deficient and adenylate cyclase-deficient cells. The evidence presented supports largely independent mechanisms of promoter induction by cyclic AMP and tax but also suggests a role for PKA-mediated phosphorylation in the regulation of HTLV-I LTR-driven gene expression by tax.

Adenylyl Cyclases↗
PubMed1991

Transcription factor AP-1 modulates the activity of the human foamy virus long terminal repeat.

The human foamy virus (HFV) contains within the U3 region of its long terminal repeat (LTR) three perfect consensus sequences for the binding of the inducible transcription factor AP-1. Results of DNase I footprint protection and gel retardation assays demonstrated that proteins in extracts of HeLa and BHK-21 cells as well as bacterially expressed Jun and Fos proteins bind to these AP-1 sites. By conducting transient expression assays using chloramphenicol acetyltransferase plasmids carrying LTR sequences with point-mutated AP-1 sites, it was found that the three AP-1 sites contribute to the optimal activity of the HFV promoter. It is shown that induction of the HFV LTR by 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum factors is mediated through the AP-1 sites.

Animals↗
PubMed1992

Nuclear protein binding to the 5' enhancer region of the intracisternal A particle long terminal repeat.

The interactions of nuclear proteins from embryonal carcinoma cells (PCC3) with the long terminal repeats (LTRs) of murine intracisternal A particle (IAP) genes were studied. Two protein-DNA complexes were detected between PCC3 nuclear extract and IAP LTRs in a gel mobility shift assay. An additional complex was observed when enriched fractions from a heparin-agarose column were used as the source of proteins. Two regions within the LTR of IAP 81 were identified as the sites of protein interaction by DNase I protection. One region encompasses 43 nucleotides within the U3 region at the 5' end of LTR 81. The other covers a 78 base pair region lying within 100 nucleotides upstream from the transcription initiation site. Studies using constructs containing intact or deleted versions of the LTR fused to the bacterial chloramphenicol acetyltransferase gene indicated that the absence of the 5' 47 base pairs reduced the level of chloramphenicol acetyltransferase transcription to 20% of that driven by the entire LTR. Southwestern analysis of PCC3 nuclear extracts and column fractions revealed that a 28,000- and a 46,000-dalton protein were the major species that interact with the 5' end of IAP LTR 81.

Animals↗
PubMed1991

Human T-cell leukemia virus (HTLV) type II Rex protein binds specifically to RNA sequences of the HTLV long terminal repeat but poorly to the human immunodeficiency virus type 1 Rev-responsive element.

The human T-cell leukemia viruses (HTLVs) encode a trans-regulatory protein, Rex, which differentially regulates viral gene expression by controlling the cytoplasmic accumulation of viral mRNAs. Because of insufficient amounts of purified protein, biochemical characterization of Rex activity has not previously been performed. Here, utilizing the baculovirus expression system, we purified HTLV type II (HTLV-II) Rex from the cytoplasmic fraction of recombinant baculovirus-infected insect cells by heparin-agarose chromatography. We directly demonstrated that Rex specifically bound HTLV-II 5' long terminal repeat RNA in both gel mobility shift and immunobinding assays. Sequences sufficient for Rex binding were localized to the R-U5 region of the HTLV-II 5' long terminal repeat and correlate with the region required for Rex function. The human immunodeficiency virus type 1 (HIV-1), has an analogous regulatory protein, Rev, which directly binds to and mediates its action through the Rev-responsive element located within the HIV-1 env gene. We demonstrated that HTLV-II Rex rescued an HIV-1JR-CSF Rev-deficient mutant, although inefficiently. This result is consistent with a weak binding activity to the HIV-1 Rev-responsive element under conditions in which it efficiently bound the HTLV-II long terminal repeat RNA.

Animals↗
PubMed1991

Spleen focus-forming virus long terminal repeat insertional activation of the murine erythropoietin receptor gene in the T3Cl-2 friend leukemia cell line.

We have characterized the structure of the erythropoietin receptor gene promoter in normal murine erythroid tissues and in Friend-induced tumor cells. Using primer extension analysis, we identified two distinct transcriptional start sites, which were located 2 base pairs apart in anemic spleens, fetal liver, Friend-induced tumoral spleens, and mouse erythro-leukemia cells. In contrast, transcription was initiated 37 base pairs upstream of the normal cap sites in T3Cl-2, a Friend virus-induced murine erythroleukemia cell line. Also, the erythropoietin receptor mRNA in T3Cl-2 was overexpressed when compared with other erythroleukemia cell lines. We found that abnormal transcription occurring in T3Cl-2 cells resulted from an erythropoietin receptor gene alteration. Indeed, one erythropoietin receptor allele was rearranged by insertion of a spleen focus-forming virus long terminal repeat within the noncoding region of the first exon, 45 bases upstream of the ATG initiation codon and in the same 5'----3' orientation. The transcription of the rearranged allele was shown to be directed from the long terminal repeat promoter, leading to a long terminal repeat-erythropoietin receptor fusion transcript, whereas the normal erythropoietin receptor allele was weakly transcribed. Such altered receptor gene activation may provide a positive pressure in the development of tumorigenic erythroleukemia.

Animals↗
PubMed1992

Activation of the human immunodeficiency virus type 1 long terminal repeat by vaccinia virus.

A variety of DNA viruses are known to activate gene expression directed by the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). In light of the proposed use of recombinant vaccinia virus for HIV-1 vaccines, evaluation of the role of vaccinia virus in HIV-1 activation is warranted. To investigate whether vaccinia virus induces HIV LTR-directed gene expression, transient expression assays in Jurkat cells persistently infected with vaccinia virus (Jvac) using plasmid DNA containing the LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were performed. CAT activity in Jvac cells was always recorded, although the level appears to fluctuate independently of virus titers. Dual intracytoplasmic staining and fluorescence-activated cell sorter analysis showed that CAT activity was expressed in the infected cells. CAT expression was not due to plasmid replication, since plasmid DNA extracted from Jvac cells 48 h after transfection was restricted only by enzymes which recognize methylated sequences, indicating a prokaryotic source for the DNA. These findings suggest that a factor(s) present in vaccinia virus-infected cells is capable of activating the LTR of HIV-1.

Cell Line↗
PubMed1996

The effects of cisplatin and methotrexate on the expression of human immunodeficiency virus type 1 long terminal repeat.

Previous work by many groups has documented the induction of HIV-LTR (human immunodeficiency virus-long terminal repeat) following exposure of cells or whole animals to ultraviolet (UV) light and other DNA damaging agents. In these experiments we set out to determine whether exposure to the cancer chemotherapeutic agents methotrexate and cisplatin had any effect on the expression of the HIV-LTR. Using HeLa cells stably transfected with a construct in which HIV-LTR drives the expression of the reporter gene chloramphenicol acetyl transferase (CAT), we demonstrated induction of HIV-LTR 24-48 h following exposure to 50 microM cisplatin. When UV exposure (10 Jm-2) was coupled with cisplatin (50 microM) treatment (which also causes DNA damage), HIV-LTR induction was additive relative to either treatment alone. Methotrexate, which depletes the medium of tetrahydrofolate and does not induce DNA damage, induced HIV-LTR at later (6-7 days) time points than cisplatin or UV treatments. When methotrexate (128 microM) and UV (10 Jm-2) treatments were combined, the agents were synergistic with regard to HIV induction. For both drugs, though, induction was not due to generalized transcriptional activation since both cisplatin and methotrexate induced a repression of total transcription as measured in nuclear run-on assays.

Chloramphenicol O-Acetyltransferase↗
PubMed1994

Severe immunodeficiency associated with a human immunodeficiency virus 1 NEF/3'-long terminal repeat transgene.

We have generated several transgenic mouse strains carrying a human immunodeficiency virus 1 (HIV-1) NEF/3' long terminal repeat (LTR) transgene under control of a T cell-specific promoter-enhancer element, showing a depletion of CD4+ T cells in the thymus and periphery. The immunological functions of the line with the most dramatic changes in lymphocyte populations, B6/338L, were analyzed in greater detail. The presence of the transgene in the heterozygous animal is associated with a dominant severe immunodeficiency. Older animals develop lymph-adenopathy and splenomegaly. CD4+CD8+ and CD4+CD8- single positive thymocytes already are depleted in these mice at the earliest stages in ontogeny, and peripheral T cells are reduced in frequency and present cell surface marker expression, which is characteristic for memory and activated T cells. The immunological response of B6/338L mice to several viral infections is also greatly impaired. Thus, the HIV-1 NEF/3' LTR as transgene in T cells can cause immunodeficiency and disease with striking similarities to a known retrovirus-induced immunodeficiency called murine AIDS (H. C. Morse III, S. K. Chattopadhyay, M. Makino, T. N. Frederickson, A. W. Hügin, and J. W. Hartley. 1992. AIDS. 6:607).

Aging↗
PubMed1990

Induction of nuclear factor-kappa B and the human immunodeficiency virus long terminal repeat by okadaic acid, a specific inhibitor of phosphatases 1 and 2A.

We have used a specific phosphatase inhibitor, okadaic acid, to examine the role of two phosphatases, PP1 and PP2A, in the induction of NF-kappa B and the long terminal repeat of the human immunodeficiency virus type 1 (HIV-LTR). Treatment of Jurkat cells with okadaic acid induced NF-kappa B in nuclear extracts. The rate of induction by okadaic acid was delayed compared to the induction of NF-kappa B by phorbol myristate acetate (PMA). The induction of NF-kappa B by okadaic acid was enhanced by cycloheximide or phytohemagglutinin (PHA). In contrast to PMA, okadaic acid appeared to induce NF-kappa B independently of protein kinase C (PKC). That the NF-kappa B induced by okadaic acid was functional was demonstrated by the marked increase in CAT activity that occurred in Jurkat, BJA-B, and U251 cells that were transfected with HIV-LTR-CAT and treated with okadaic acid. The increase in CAT activity triggered by okadaic acid was dependent on the presence of the NF-kappa B sites in the long terminal repeat of HIV as assessed by deletion and mutation analysis. Similarly to its effect on the induction of NF-kappa B, PHA added together with okadaic acid resulted in a further increase in CAT activity. Somewhat surprisingly, the addition of PMA inhibited the increase in CAT activity in response to okadaic acid, which suggests that the activation of PKC may also induce inhibitory factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed2001

Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression.

Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners.

Amino Acids↗
PubMed1997

Activation of human immunodeficiency virus long terminal repeat by arachidonic acid.

Arachidonic acid is the precursor of highly reactive mediators, including prostaglandins and leukotrienes, and the most abundant n-6 polyunsaturated fatty acid in mammalian cell membranes. It is released from phospholipids upon many inflammatory stimuli. In this study, a chloramphenicol acyltransferase reporter gene, under control of the human immunodeficiency virus-1 long terminal repeat, was strongly induced upon treating human promonocytes with arachidonic acid. The n-3 fatty acid eicosapentenoic, found in abundance in fish oil, had no effect. HIV-1 long terminal repeat activation by arachidonic acid was suppressed by inhibitors of both lipoxygenase and cyclooxygenase pathways, suggesting that metabolites, rather than arachidonic acid itself, mediated the stimulatory effect. This is the first report linking HIV-1 expression to the metabolism of arachidonic acid.

Analysis of Variance↗
PubMed1997

Induction of transcription from the long terminal repeat of the intracysternal particles type A (IAP) by X-irradiation.

Intracisternal A particles (IAPs) are retrovirus-like entities that are present in many embryonic and transformed cells of Mus musculus. They present long terminal repeats (LTRs) which control the promotion and regulation of their transcription. Using a construction expressing a reporter gene under the control of the entire long terminal repeat (LTR) of IAP in transfected murine fibroblast BALB/c 3T3 cells clone D152, we were able to show that the IAP-LTR is activated by X-irradiation in a time-dependent manner. The relative CAT activity increased with increasing X-irradiation doses, reaching a maximum at 75-150 cGy, followed by a drop in activation. In addition, X-induced D152 mouse cells produced extracellular factor(s), in response to X-irradiation, which activated the IAP-LTR in non-irradiated cells. This factor(s) was detected both when transfected cells were cocultured with inducing cells and when conditioned medium from irradiated cultures was added to the cell cultures. The use of suramin, a strong polyanonic molecule which has been reported to trap growth factors, induces a high reduction of the indirect activation.

3T3 Cells↗
PubMed1994

Transcription factor AP-4 participates in activation of bovine leukemia virus long terminal repeat by p34 Tax.

Three 21bp repeats can be found in the bovine leukemia virus long terminal repeat, which are crucial for the LTR directed gene expression by the trans activator protein Tax. Previous studies demonstrated that the major target of the Tax directed activation are the CRE-like elements in the center of these repeats. In this work we report that another motif of the 21bp repeats is also required for the Tax activation. Gel retardation--with the wild type or mutant 21bp repeats--revealed that cellular factors from HeLa cells were specifically bound to the center (CRE-like element) and the 3' region of the repeats, which contains a CAGCTG consensus AP-4 binding site. In vivo analysis using the synthetic 21bp repeats indicated that beyond the consensus CRE-like motif, the AP-4 site is also essential for Tax activation. To determine the role of AP-4 in BLV Tax trans activation, we used the AP-4 cDNA in antisense transient assays. In the in vivo experiments the antisense AP-4 RNA resulted in strongly decreased Tax activation. On the basis of these results we conclude that AP-4 is a good candidate of cellular factors involved in BLV Tax trans activation.

Animals↗
PubMed1995

Role of glucocorticoid receptor binding sites in the human immunodeficiency virus type 1 long terminal repeat in steroid-mediated suppression of HIV gene expression.

Dexamethasone inhibited human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-directed gene expression in cells of T and B lymphoblastoid lineages, but not in monocytic cells. Suppression required an intact glucocorticoid receptor (GR), as it was amplified by transfection of lymphocytes with a plasmid encoding the human GR and blocked by the receptor antagonist RU486. These results were in direct contrast to the effects of dexamethasone on a murine leukemia retrovirus promoter where, consistent with the findings of others, activation of gene expression was obtained. Potential regions of the HIV-1 LTR mediating these effects were sought, with sequence homologies predicting two new glucocorticoid response element half-sites, GRE-II (nucleotides -6 to -1) and GRE-III (+ 15 to + 20), downstream from a previously identified GR DNA binding domain, GRE-I (-264 to -259). Mutational analyses documented the loss of inhibitory activity attendant on changes in GRE-III and the independence of steroid-mediated effects from GRE-I and GRE-II. Consistent with these findings, electrophoretic mobility shift assays revealed a difference in binding of cellular factors to GRE-III in cells of T and B lymphocyte vs. monocytic lineages. Binding sites for the cellular transcription factor leader binding protein (LBP-1) were found to overlap with GRE-III, and LBP-1 interacted with this element in the HIV LTR only in T and B lymphocytic extracts. We hypothesize that GRE-III sequence-specific effects, including modulation of LBP-GR interactions, underlie the negative regulatory effect of glucocorticoids on HIV-1 gene expression, with some specificity for cell type.

Base Sequence↗