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PubMed1983

Elements in the long terminal repeat of murine retroviruses enhance stable transformation by thymidine kinase gene.

We have investigated the effects of long terminal repeats (LTRs) of murine retroviruses on the frequency of obtaining stable transfectants by the herpes virus thymidine kinase (TK) gene. The results indicate that addition of LTRs enhances the number of TK+ transformants by 10-20 fold. A 5-12 fold enhancement was also observed when chromosomal DNA from either human or hamster cells was mixed with a plasmid containing LTR sequences and transfected onto LTR- cells. The LTR sequences involved in the enhancement were localized in the region which contains tandem repeats. All other regions of the LTR did not show any enhancement of stable TK+ transfectants. The location or the orientation of the enhancer sequences with respect to the TK gene did not exert any influence on the frequency of transformation. The enhancement effect does not appear to be linked to either increased numbers of chromosomal integrations or elevated levels of transcription of the TK gene.

Animals↗
PubMed1985

Bovine leukemia virus long terminal repeat: a cell type-specific promoter.

The functional activity of the promoter unit contained within the long terminal repeat (LTR) of bovine leukemia virus (BLV) was examined by monitoring transient expression of a heterologous gene placed under its control. Various cell lines were transfected with recombinant plasmids carrying the bacterial chloramphenicol acetyltransferase (CAT) gene coupled to the BLV LTR (pBL-cat). Transient expression of CAT activity directed by the BLV LTR was observed only in the established BLV-producer cell lines derived from fetal lamb kidney (FLK) cells and bat lung cells. The amount of CAT activity transiently expressed in FLK-BLV cells was decreased approximately tenfold by deletion of LTR sequences located within a region 100 to 170 nucleotides upstream of the RNA start site. Surprisingly, removal of the region 50 base pairs downstream of the RNA initiation site to the 3'-end of the LTR reduced the expression of CAT activity by 87 percent. The BLV LTR thus appears to be an unusual promoter unit, functioning in a cell type-specific manner and possessing sequences on both the 5' and 3' sides of the RNA start site that influence gene expression.

Animals↗
PubMed1985

Tissue selectivity of murine leukemia virus infection is determined by long terminal repeat sequences.

Here we show that the tissue specificity of murine retrovirus infections is determined by the long terminal repeat (LTR) of an otherwise isogenic set of viruses. The isogenic viruses used for this study contain the coding gag, pol, and env genes of the avirulent Akv virus. Recombinant viruses that contain the LTR of a virus that induces T-cell leukemia lymphoma preferentially infect T lymphocytes. Viruses that carry the LTR of a virus that induces erythroleukemia preferentially infect non-T lymphoblastoid cell lines in the marrow and spleen. The Akv virus itself displays no tissue preference for hematopoietic cells. These experiments suggest that retroviruses that carry appropriate enhancer-promoters can be used to infect selectively specific target cells in animals.

Animals↗
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↗
PubMed1991

Identification of a novel mammary cell line-specific enhancer element in the long terminal repeat of mouse mammary tumor virus, which interacts with its hormone-responsive element.

We analyzed the long terminal repeat (LTR) of mouse mammary tumor virus for sequences that influence its promoter activity by using the chloramphenicol acetyltransferase assay. A series of LTR deletion mutants and recombinants between LTR and simian virus 40 regulatory sequences were used for these studies. Through transfection experiments in three different human cell lines (T47D, MCF-7, and HeLa), we identified a novel mammary cell line-specific enhancer element on a 98-bp BanII fragment (from position -1075 to -978 upstream of the start site of transcription) which interacts with the hormone-responsive element of LTR. We also identified nuclear factors that specifically interacted with this BanII fragment in the nuclear extract from the mammary tumor cell line, T47D, but not from the HeLa cell line.

Breast Neoplasms↗
PubMed1994

Identification of a region of a murine leukemia virus long terminal repeat with novel transcriptional regulatory activities.

The 93-bp region downstream of the enhancer (DEN) in the long terminal repeat (LTR) of the mink cell focus-forming virus (MCF13) has been shown to be important for transcriptional activation and viral lymphomagenicity (J. C. Tupper, H. Chen, E. F. Hays, G. C. Bristol, and F. K. Yoshimura, J. Virol. 66:7080-7088, 1992). In this report, we have further explored the role of the DEN region in transcriptional activation. We observed that it has enhancer-like abilities as well as some unique LTR properties. Transcriptional activation by the DEN region involved interactions with enhancer sequences that were either synergistic or additive, depending on the cell type. The most intriguing property of the DEN region is its ability to induce transcription in activated T cells. This activity is unique for the LTR in that no other LTR region can do this. We also examined the role of the DEN region in retroviral lymphomagenesis. We cloned and sequenced proviral LTRs integrated upstream of the cellular c-myc gene from DNA obtained from thymic tumors induced by DEN region deletion mutant viruses in AKR mice. We determined that for transcriptional activation of the c-myc proto-oncogene, enhancer sequences can substitute for the DEN region. This study identifies the significance of non-enhancer sequences in the LTR for the oncogenesis of the MCF13 retrovirus.

Animals↗
PubMed1993

The NF-kappa B and Sp1 motifs of the human immunodeficiency virus type 1 long terminal repeat function as novel thyroid hormone response elements.

We report that thyroid hormone (T3) receptor (T3R) can activate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Purified chick T3R-alpha 1 (cT3R-alpha 1) binds as monomers and homodimers to a region in the LTR (nucleotides -104 to -75 [-104/-75]) which contains two tandem NF-kappa B binding sites and to a region (-80/-45) which contains three Sp1 binding sites. In contrast, human retinoic acid receptor alpha (RAR-alpha) and mouse retinoid X receptor beta (RXR-beta) do not bind to these elements. However, RXR-beta binds to these elements as heterodimers with cT3R-alpha 1 and to a lesser extent with RAR-alpha. Gel mobility shift assays also revealed that purified NF-kappa B p50/65 or p50/50 can bind to one but not both NF-kappa B sites simultaneously. Although the binding sites for p50/65, p50/50, and T3R, or Sp1 and T3R, overlap, their binding is mutually exclusive, and with the inclusion of RXR-beta, the major complex is the RXR-beta-cT3R-alpha 1 heterodimer. The NF-kappa B region of the LTR and the NF-kappa B elements from the kappa light chain enhancer both function as T3 response elements (TREs) when linked to a heterologous promoter. The TREs in the HIV-1 NF-kappa B sites appear to be organized as a direct repeat with an 8- or 10-bp gap between the half-sites. Mutations within the NF-kappa B motifs which eliminate binding of cT3R-alpha 1 also abolish stimulation by T3, indicating that cT3R-alpha 1 binding to the Sp1 region does not independently mediate activation by T3. The Sp1 region, however, is converted to a functionally strong TRE by the viral tat factor. These studies indicate that the HIV-1 LTR contains both tat-dependent and tat-independent TREs and reveal the potential for T3R to modulate other genes containing NF-kappa B- and Sp1-like elements. Furthermore, they indicate the importance of other transcription factors in determining whether certain T3R DNA binding sequences can function as an active TRE.

Base Sequence↗
PubMed1994

Stably integrated mouse mammary tumor virus long terminal repeat DNA requires the octamer motifs for basal promoter activity.

In the mouse mammary tumor virus promoter, a tandem of octamer motifs, recognized by ubiquitous and tissue-restricted Oct transcription factors, is located upstream of the TATA box and next to a binding site for the transcription factor nuclear factor I (NF-I). Their function was investigated with mutant long terminal repeats under different transfection conditions in mouse Ltk- cells and quantitative S1 nuclease mapping of the transcripts. In stable transfectants, which are most representative of the state of proviral DNA with respect to both number of integrated DNA templates and chromatin organization, a long terminal repeat mutant of both octamer sites showed an average 50-fold reduction of the basal transcription level, while the dexamethasone-stimulated level was unaffected. DNase I in vitro footprinting assays with L-cell nuclear protein extracts showed that the mutant DNA was unable to bind octamer factors but had a normal footprint in the NF-I site. I conclude that mouse mammary tumor virus employs the tandem octamer motifs of the viral promoter, recognized by the ubiquitous transcription factor Oct-1, for its basal transcriptional activity and the NF-I binding site, as previously shown, for glucocorticoid-stimulated transcription. A deletion mutant with only one octamer site showed a marked base-level reduction at high copy number but little reduction at low copies of integrated plasmids. The observed transcription levels may depend both on the relative ratio of transcription factors to DNA templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting.

Animals↗
PubMed2002

Tat and trans-activation-responsive (TAR) RNA-independent induction of HIV-1 long terminal repeat by human and murine cyclin T1 requires Sp1.

P-TEFb, cyclin T1 + CDK9, is needed for the expression of cellular promoters and primate lentiviral long terminal repeats (LTRs). Curiously, cellular and lentiviral promoters differ dramatically in the requirements for positive transcriptional elongation factor (P-TEF) b activity. Lentiviral LTRs, but not cellular promoters, need an RNA-associated P-TEFb/Tat/TAR (trans-activation-responsive) RNA ternary complex. Ternary complex defective murine cycT1 is apparently inactive for lentiviral transcription. Why P-TEFb requires Tat/TAR for LTRs but not for cellular promoters remains unknown. To explore this question, we sought to determine whether DNA targeting of murine and human cyclin T1 can reconstitute a Tat/TAR-independent activity to the HIV-1 LTR. In the absence of Tat and TAR, we found that both HuCycT1 and MuCycT1 can robustly activate the HIV-1 LTR. We further showed that Sp1 is necessary and sufficient for this DNA-targeted activity. Thus, like cellular promoters, HIV-1 LTR can use P-TEFb function without a Tat/TAR RNA complex. This activity could explain recent findings of robust HIV-1 replication in rat cells that cannot form a P-TEFb/Tat/TAR moiety.

Animals↗
PubMed1997

Sequence elements downstream of the human immunodeficiency virus type 1 long terminal repeat are required for efficient viral gene transcription.

We investigated the role of a 54-nucleotide region (+200 to +253) located downstream of the HIV-1 long terminal repeat (LTR) on virus gene expression and found, using RT-PCR and p24 CA analysis, that deletion of this region inhibited synthesis of both viral RNA and protein. CAT assays showed that these results were attributable to decreased transcription efficiency of the HIV-1 LTR and not to the stability of the RNA transcripts produced. Further deletional analysis and transfection studies showed that the most important sequences with regard to proviral DNA expression were located between nucleotide positions +218 and +237. Furthermore, substitutional mutational analysis showed that a CTCTCTC sequence at positions +227 to +233, homologous to the pyrimidine-rich initiator (Inr) region found in several promoters, was required for efficient production of both viral RNA and protein. Deletion of the sequence +200 to +217, homologous to the interferon-stimulated response element (ISRE), resulted in impaired LTR promoter activity and decreased synthesis of viral RNA and protein. However, when the latter region was replaced by homologous ISRE sequences from an interferon-stimulated gene (ISG-54), an even more severe effect on HIV gene expression and replication was observed, suggesting that ISRE-like sequences in HIV act differently from homologous sequences in interferon-responsive cellular genes.

Animals↗
PubMed1995

The human immunodeficiency virus long terminal repeat includes a specialised initiator element which is required for Tat-responsive transcription.

The effects of mutations in human immunodeficiency virus type-1 (HIV-1) long terminal repeat on initiation and on Tat-mediated trans-activation were studied using cell-free transcription assays. All the elements that are necessary for efficient transcription initiation in vitro are included in the core promoter. This region contains three tandem Sp1 binding sites, a TATA element and an initiator (INR) sequence. Although the HIV-1 INR element overlaps the trans-activation response region (TAR), it forms an integral part of the promoter. The HIV-1 INR element was characterised in detail using a template that carries a complete HIV-1 promoter and a displaced TAR RNA element. The results demonstrate that the sequence G+1GGTCT is essential for HIV-1 INR function. RNase protection experiments show that Tat acts exclusively to stimulate transcriptional elongation. Mutations in the core promoter elements reduce initiation rates dramatically but do not block Tat activity. For each mutation studied, the total level of transcription in the presence of Tat is proportional to the rate of initiation in the absence of Tat. Furthermore the rate of initiation remains constant in the presence or absence of Tat. We conclude that the elements of the HIV-1 core promoter act in concert to simulate initiation. By contrast, Tat acts independently of the core promoter elements and stimulates elongation. The data strongly suggest that Tat is recruited to the elongating transcription complex during its transit through TAR.

Base Sequence↗
PubMed2005

Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-kappaB pathway and functionally cooperates with Tat.

BACKGROUND: The nuclear transcription factor NF-kappaB binds to the HIV-1 long terminal repeat (LTR) and is a key regulator of HIV-1 gene expression in cells latently infected with this virus. In this report, we have analyzed the ability of Kaposi's sarcoma associate herpes virus (KSHV, also known as Human Herpes virus 8)-encoded viral FLIP (Fas-associated death domain-like IL-1 beta-converting enzyme inhibitory protein) K13 to activate the HIV-1 LTR. RESULTS: We present evidence that vFLIP K13 activates HIV-1 LTR via the activation of the classical NF-kappaB pathway involving c-Rel, p65 and p50 subunits. K13-induced HIV-1 LTR transcriptional activation requires the cooperative interaction of all three components of the IKK complex and can be effectively blocked by inhibitors of the classical NF-kappaB pathway. K13 mutants that lacked the ability to activate the NF-kappaB pathway also failed to activate the HIV-1 LTR. K13 could effectively activate a HIV-1 LTR reporter construct lacking the Tat binding site but failed to activate a construct lacking the NF-kappaB binding sites. However, coexpression of HIV-1 Tat with K13 led to synergistic activation of HIV-1 LTR. Finally, K13 differentially activated HIV-1 LTRs derived from different strains of HIV-1, which correlated with their responsiveness to NF-kappaB pathway. CONCLUSIONS: Our results suggest that concomitant infection with KSHV/HHV8 may stimulate HIV-1 LTR via vFLIP K13-induced classical NF-kappaB pathway which cooperates with HIV-1 Tat protein.

Animals↗
PubMed2004

Unusually long target site duplications flanking some of the long terminal repeats of human endogenous retrovirus K in the human genome.

Human endogenous retroviruses (HERVs) make up a substantial part of the human genome. HERVs and solitary long terminal repeats (solo LTRs) are usually flanked by 4-6 nt short direct repeats through the well-known mechanism of their integration. A number of solo LTRs flanked by unusually long direct repeats were detected in the human genome. These unusual structures might be a product of an alternative virus insertion mechanism.

Endogenous Retroviruses↗
PubMed1991

DNA CpG methylation inhibits binding of NF-kappa B proteins to the HIV-1 long terminal repeat cognate DNA motifs.

The regulation of cellular or viral gene expression is directly influenced by the pattern of methylated cytosine residues localized in the DNA of enhancer/promoter sequences. The mechanism of transcriptional silencing has been explained on the basis of either an indirect model, in which densely methylated DNA is recognized by proteins that may displace crucial transcription factors, or a direct model, in which binding of a single transcription protein is prevented by the presence of a methylated CpG dinucleotide localized in a sensitive region of a DNA motif. In this study, we have determined that methylation of the core CpG dinucleotide located within the NF-kappa B repeated motifs of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat can inhibit the binding of the NF-kappa B protein complex from crude nuclear extracts or from purified bovine spleen and specifically inhibit the binding of recombinant p50 protein. We have used the electrophoretic mobility shift assay (EMSA) and DNaseI footprinting analysis to demonstrate that binding of the NF-kappa B proteins to their cognate motifs can be inhibited via the direct model proposed for methylation-mediated inhibition of DNA-protein interaction.

5-Methylcytosine↗
PubMed1993

Twenty-one base pair repeat elements influence the ability of a Gal4-Tax fusion protein to transactivate the HTLV-I long terminal repeat.

The Tax1 protein of the human T-cell leukemia virus (HTLV-I) is a 40-kDa positive transactivator of viral gene expression. Tax1 does not bind directly to DNA, but associates indirectly with DNA via cellular transcription factors. To further investigate the activation of HTLV-I transcription by Tax1, a chimeric protein containing Tax1 fused to the DNA binding domain of Gal4 was created (Gal4-Tax). HTLV-I long terminal repeat (LTR) reporter plasmids were constructed in which specific Tax1 responsive elements were replaced with Gal4 binding sites. Cotransfection of Gal4-Tax or Tax1 with HTLV-I LTR reporter constructs containing Gal4 binding sites demonstrated that Gal4 sequences were necessary but not sufficient for maximal activation of the promoter by Gal4-Tax. Sequences surrounding the Gal4 binding sites were important in determining the level of Gal4-Tax activation. Association of Gal4-Tax with promoters which contained six Gal4 binding sites, but which lacked flanking LTR sequences, were weakly transactivated by Gal4-Tax (sevenfold). In contrast, LTR-CAT reporter constructs containing three Gal4 binding sites flanked by two 21 base pair repeat elements demonstrated a ninefold greater response to Gal4-Tax. These results suggest that cellular transcription factors, which bind the 21 base pair repeat elements, influence the ability of Tax1 to function as a transactivator. Furthermore, this effect is not fully explained by the ability of these factors to physically direct Tax1 to the LTR.

Amino Acid Sequence↗
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↗
PubMed1994

Activation of the human immunodeficiency virus long terminal repeat in THP-1 cells by a staphylococcal extracellular product.

Staphylococcal strains can release a factor that strongly activates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in THP-1 cells transfected with the HIV-1 LTR-driven luciferase reporter gene (THP-1 LTRluc). The factor is present in the overnight culture fluid and is readily released from the organisms into aqueous medium by vigorous mixing. Staphylococcal extracellular material is a complex mixture of polysaccharide and protein containing peptidoglycan and teichoic acid, released in part by cell wall turnover. The importance of the carbohydrate component is emphasized by concanavalin A (Con A) inhibition of staphylococcal product-induced LTR activation but not of activation by phorbol 12-myristate 13-acetate or tumor necrosis factor. The effect of Con A was decreased or abolished by sugars in the order methyl alpha-D-mannopyranoside > methyl alpha-D-glucopyranoside > mannose > glucose = fructose > N-acetylglucosamine. Wheat germ agglutinin was less inhibitory than Con A; in this instance N-acetylglucosamine decreased inhibition, whereas methyl alpha-D-mannopyranoside or methyl alpha-D-glucopyranoside did not. The induction of luciferase activity in THP-1 LTRluc by the staphylococcal extracellular product also was inhibited by fetal bovine and normal human serum. A comparison of 31 staphylococcal isolates (9 Staphylococcus aureus, 11 Staphylococcus epidermidis, 2 Staphylococcus haemolyticus, 4 Staphylococcus hominis, 2 Staphylococcus capitis, 2 Staphylococcus warneri, 1 Staphylococcus saprophyticus) revealed wide variation in LTR activating activity that did not correlate closely with slime production. Our findings, using induction of luciferase in THP-1 LTRluc as a model for upregulation of HIV infection, raise the possibility that staphylococci, as well as certain other microorganisms, release carbohydrate-containing exopolymers, which can activate the HIV-1 LTR, thus influencing progression of HIV infection.

Animals↗