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PubMed2004

Tat-dependent repression of human immunodeficiency virus type 1 long terminal repeat promoter activity by fusion of cellular transcription factors.

Transcription initiation from HIV-1 long terminal repeat (LTR) promoter requires the virally encoded transactivator, Tat, and several cellular co-factors to accomplish the Tat-dependent processive transcription elongation. Individual cellular transcription activators, LBP-1b and Oct-1, on the other hand, have been shown to inhibit LTR promoter activities probably via competitive binding against TFIID to the TATA-box in LTR promoter. To explore the genetic interference strategies against the viral replication, we took advantage of the existence of the bipartite DNA binding domains and the repression domains of LBP-1b and Oct-1 factors to generate a chimeric transcription repressor. Our results indicated that the fusion protein of LBP-1b and Oct-1 exhibited higher DNA binding affinity to the viral promoter than the individual factors, and little interference with the host cell gene expression due to its anticipated rare cognate DNA sites in the host cell genome. Moreover, the chimera exerted increased Tat-dependent repression of transcription initiation at the LTR promoter both in vitro and in vivo compared to LBP-1b, Oct-1 or combination of LBP-1b and Oct-1. These results might provide the lead in generating a therapeutic reagent useful to suppress HIV-1 replication.

DNA-Binding Proteins↗
PubMed1990

Transcriptional activation of the human immunodeficiency virus long terminal repeat sequences by cis-platin.

We constructed a recombinant plasmid, pBHIV1 carrying the long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1), linked to the chloramphenicol acetyl transferase (CAT) gene plasmid. Plasmid pBHIV1 also contains the aminoglycoside phosphotransferase gene as a selectable marker. We introduced pBHIV1 in rat 208F fibroblasts and obtained stable geneticin resistant RFBHIV1-1 transfectant cells. A further control used was plasmid p202A, which carries the mutant T24 H-ras1 promoter linked to the promotorless cat gene. Plasmid p202A also carries the aph gene as a selectable marker and was transfected into 208F cells to obtain stable transfectant RF202A-1 cells. Both RFBHIV1-1 and RF202A-1 cells expressed CAT activity from the HIV LTR and T24 H-ras1 promoters. The response to cis-platin, a platin derivative and hexadecyl-phosphocholine was studied on the HIV LTR and H-ras1 regulated CAT activity in RFBHIV1-1 and RF202A-1 cells. It was found that at 5 x 10(-5) M concentrations cis-platin stimulates by 22-fold the expression of CAT from the HIV LTR, whereas only a 4-fold stimulation was observed on the T24 H-ras1 promoter. Our results suggest caution against therapy including this compound at cytotoxic concentrations in the treatment of AIDS patients.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Human immunodeficiency virus type 1 subtypes have a distinct long terminal repeat that determines the replication rate in a host-cell-specific manner.

The long terminal repeat (LTR) transcriptional promoters of different human immunodeficiency virus (HIV) type 1 subtypes were inserted into the LAI molecular clone of subtype B. The viral genotypes represent seven subtypes (A, B, C, D, E, F, and G) and one circulating recombinant form (AG). We performed replication studies with this isogenic set of viruses across six cellular environments. This approach revealed strong cellular environment effects, but the method was not sensitive enough to detect small differences in the replication rate between the subtypes. By conducting pairwise competition experiments between the virus variants in six cellular environments, we could demonstrate significant differences in the replication rates of the subtypes and that LTR-determined viral fitness depends both on the host cell type and the activation state of the cell. In addition, we determined the degree of conservation of the transcription factor-binding sites (TFBS) in the different-subtype LTRs by analyzing sequences from the HIV sequence database. The sequence analyses revealed subtype-specific conservation of certain TFBS. The results indicate that one should consider the possibility of subtype-specific viral replication rates in vivo, which are strongly influenced by the host environment. We argue that the multidimensional host environment may have shaped the genetic structures of the subtype LTRs.

Cell Division↗
PubMed1993

BEL-1 transactivator responsive sequences in the long terminal repeat of human foamy virus.

Cis-regulatory elements in the long terminal repeat (LTR) of human foamy virus (HFV) were identified by using LTR mutants to transiently express the chloramphenicol acetyl-transferase gene after co-transfection with an expression plasmid for the virus bel-1 (transactivator) gene. The R-U5 region and an element in the 5' U3 region were found to negatively influence HFV gene expression. The complete BEL-1 responsive region was mapped to extend from nucleotide position -471 to position -93 relative to the start of transcription. Within this region, three elements were identified that in the homologous or a heterologous (SV40) promoter context can, independently and irrespective of their orientation, act as targets for BEL-1. These elements are located between nucleotide positions -413/-378, -361/-291, and -124/93. The target elements do not share obvious sequence homologies. The mechanism of HFV transactivation appears to be novel among the complex retroviruses and is likely to involve, as yet, undiscovered cellular DNA binding factors.

Animals↗
PubMed1994

Structure and function of the long terminal repeat of the chimpanzee foamy virus isolates (SFV-6).

The complete long terminal repeat (LTR) nucleotide sequence of the chimpanzee foamy virus isolate SFV-6 was determined. Its 1761-bp size makes it the longest LTR reported to date among all retroviruses. Since the length of its LTR is similar to that of other simian isolates while its sequence homology is closer to that of HFV, SFV-6 genetic structure appears to be intermediate between simian and human foamy viruses. Transient expression assays demonstrate that SFV-6 encodes a transactivator of viral gene expression directed either by its own LTR or by heterologous promoters like HFV and HIV-1 LTRs. Our data also provide evidence for cross-transactivation between SFV-6 and HFV.

Animals↗
PubMed2005

Leukemia virus long terminal repeat activates NFkappaB pathway by a TLR3-dependent mechanism.

The long terminal repeat (LTR) region of leukemia viruses plays a critical role in tissue tropism and pathogenic potential of the viruses. We have previously reported that U3-LTR from Moloney murine and feline leukemia viruses (Mo-MuLV and FeLV) upregulates specific cellular genes in trans in an integration-independent way. The U3-LTR region necessary for this action does not encode a protein but instead makes a specific RNA transcript. Because several cellular genes transactivated by the U3-LTR can also be activated by NFkappaB, and because the antiapoptotic and growth promoting activities of NFkappaB have been implicated in leukemogenesis, we investigated whether FeLV U3-LTR can activate NFkappaB signaling. Here, we demonstrate that FeLV U3-LTR indeed upregulates the NFkappaB signaling pathway via activation of Ras-Raf-IkappaB kinase (IKK) and degradation of IkappaB. LTR-mediated transcriptional activation of genes did not require new protein synthesis suggesting an active role of the LTR transcript in the process. Using Toll-like receptor (TLR) deficient HEK293 cells and PKR(-/-) mouse embryo fibroblasts, we further demonstrate that although dsRNA-activated protein kinase R (PKR) is not necessary, TLR3 is required for the activation of NFkappaB by the LTR. Our study thus demonstrates involvement of a TLR3-dependent but PKR-independent dsRNA-mediated signaling pathway for NFkappaB activation and thus provides a new mechanistic explanation of LTR-mediated cellular gene transactivation.

3T3 Cells↗
PubMed1985

The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat.

The location of cis-acting regulatory sequences within the long terminal repeat (LTR) of the human T cell lymphotropic virus type III (HTLV-III/LAV) was determined. An enhancer element capable of increasing the rate of transcription from a heterologous promoter, irrespective of distance and orientation, is located between nucleotides -137 and -17 (cap site +1). The promoter sequences present near the TATA box respond to heterologous enhancers. The sequences present between nucleotides -17 and +80 are responsive to HTLV-III-associated trans-acting regulatory factors. Activation of these sequences by the viral regulatory factors requires the presence of a functional enhancer. The enhancer requirement is nonspecific, as the enhancer sequences of RSV, HTLV-I, and SV40 can functionally replace the HTLV-III enhancer. These findings define a new type of regulatory element, provide insight into the mechanisms that regulate HTLV-III gene expression, and may help to explain the effects of this virus on infected cells.

Acetyltransferases↗
PubMed1985

Location of cis-acting regulatory sequences in the human T-cell leukemia virus type I long terminal repeat.

The location of cis-acting regulatory regions within the long terminal repeat (LTR) of the human T-cell leukemia virus type I (HTLV-1) was determined. The sequences present between nucleotides -350 and -55 (cap site +1) contain an enhancer element that is active in lymphoid and nonlymphoid cell lines. The sequences located near the "TATA" and RNA initiation sites contain a promoter, the activity of which can be augmented by homologous and heterologous enhancer elements. A region responsive to trans-acting transcription factors present in HTLV-I- and HTLV type II-infected cells is located between nucleotides -159 and +315. HTLV-I LTR deletion mutants respond in a similar manner both to the trans-acting factors present in infected cells and to the tat protein encoded by the x-lor region of the genome, thus providing further evidence that the tat protein mediates transcriptional trans-activation of the LTR in HTLV-infected cells.

Avian Sarcoma Viruses↗
PubMed1985

Characterization of long terminal repeat sequences of HTLV-III.

The nucleotide sequence of the long terminal repeat sequence (LTR) of the human T-cell leukemia (lymphotropic) virus type III (HTLV-III) was determined. This virus is associated etiologically with the acquired immune deficiency syndrome. The LTR was found to be 634 base pairs in length with U3, R, and U5 regions of 453, 98, and 83 bp, respectively. The proviral DNA is flanked by a 7-base-pair direct repeat. The promoter and polyadenylation signals are situated 27 and 24 base pairs upstream from the respective transcriptional initiation and polyadenylation sites. The primer binding site is complementary to transfer RNA-lysine. The LTR of HTLV-III, like that of HTLV-I, showed a limited homology to enhancer-like sequences within two genes expressed specifically in T lymphocytes, T-cell growth factor, and gamma-interferon. Structural comparisons revealed that the LTR of HTLV-III is distantly related to those of HTLV-I, HTLV-II, and bovine leukemia virus.

Biological Evolution↗
PubMed2000

Target specificity of the endonuclease from the Xenopus laevis non-long terminal repeat retrotransposon, Tx1L.

Elements of the Tx1L family are non-long terminal repeat retrotransposons (NLRs) that are dispersed in the genome of Xenopus laevis. Essentially all genomic copies of Tx1L are found inserted at a specific site within another family of transposable elements (Tx1D). This suggests that Tx1L is a site-specific retrotransposon. Like many (but not all) other NLRs, the Xenopus element encodes an apparent endonuclease that is related in sequence to the apurinic-apyrimidinic endonucleases that participate in DNA repair. This enzyme is thought to introduce the single-strand break in target DNA that initiates transposition by the target-primed reverse transcription (TPRT) mechanism. To explore the issue of target specificity more fully, we expressed the polypeptide encoded by the endonuclease domain of open reading frame 2 from Tx1L (Tx1L EN) and characterized its cleavage capabilities. This endonuclease makes a specific nick in the bottom strand precisely at one end of the presumed Tx1L target duplication. Because this activity leaves a 5'-phosphate and 3'-hydroxyl at the nick, it has the location and chemistry required to initiate new insertion events by TPRT. Tx1L EN does not make a specific cut at a preferred target site for Tx1D elements, ruling out the alternative possibility that the composite Tx1L-Tx1D element moves as a unit under the control of functions encoded by Tx1L. Further characterization revealed that the endonuclease remains active for many hours at room temperature and that it is capable of enzymatic turnover. Scanning substitution mutagenesis located the recognition site for Tx1L EN within 10 bp surrounding the primary nick site. Implications of these features for natural transposition events are discussed.

Animals↗
PubMed1989

Isolation and characterization of a protein fraction that binds to enhancer core sequences in intracisternal A-particle long terminal repeats.

The U3 region of mouse intracisternal A-particle (IAP) long terminal repeats (LTRs) contains several nuclear protein-binding domains. Two of these contain sequences with homology to the SV40 enhancer core. We refer to these two domains as Enh1 and Enh2. The Enh2 domain is an important determinant of promoter activity in vivo. We report here the isolation of nuclear fractions from human 293 and mouse MOPC-315 cells which interact with Enh1 and Enh2. Purification was achieved via DNA-affinity chromatography on a multimerized oligonucleotide representing the Enh2 region from the LTR of the mouse genomic IAP element, MIA14. Glycerol gradient sedimentation suggested a native Mr of approximately 80-100 for the binding component(s) in both crude and affinity-purified fractions. UV cross-linking showed that the binding activity involved two polypeptides within this size range. The affinity-isolated fraction from each cell line was highly purified, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in vitro binding analysis. Exonuclease III footprinting showed that the two polypeptides interacted preferentially with the Enh1 and Enh2 domains within a 139-base pair segment from the MIA14 LTR. The polypeptides interacted in a sequence-specific manner with oligonucleotides representing these domains within the IAP LTR and with oligonucleotides containing the enhancer core sequence from SV40 and polyoma virus. Equilibrium binding studies indicated that the apparent dissociation constants for the polypeptides binding to the enhancer core sequence from MIA14, SV40, and polyoma virus were similar. Therefore, this affinity-purified fraction may represent a novel enhancer core-binding component which is distinct from the previously characterized rat CCAAT/enhancer-binding protein, C/EBP.

Animals↗
PubMed1993

Identification of transactivation-response sequences in the long terminal repeat of bovine immunodeficiency-like virus.

Transient expression assays using the reporter gene that encodes chloramphenicol acetyltransferase were used to identify cis-acting sequences necessary for bovine immunodeficiency-like virus (BIV) transactivation. Computer analyses identified two RNA stem-loop structures located immediately downstream of the transcription start site in the long terminal repeat. Deletion analysis of the long terminal repeat indicated that sequences containing the proximal stem-loop structure located between +4 and +31 are required for virus-specific transactivation. Therefore, BIV likely utilizes a mechanism of transactivation similar to that of the human and simian lentiviruses.

Animals↗
PubMed1993

NF-IL6-mediated transcriptional activation of the long terminal repeat of the human immunodeficiency virus type 1.

An upstream control region in the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) includes a potential negative regulatory element (NRE1). Cotransfecting multimers of a sequence spanning this element with an LTR-CAT construct produced an increase in chloramphenicol acetyltransferase (CAT) activity in Jurkat and HepG2 cells, providing further evidence and support for the existence of an NRE. In screening experiments aimed at identifying those factors that regulate HIV-1 transcription through interactions with the NRE1 region, we isolated a cDNA for NF-IL6. Previous studies have shown that NF-IL6 is a key nuclear factor that activates gene expression in response to interleukin 6. By methylation interference analysis, we have localized the NF-IL6 binding site within the NRE1 region and found that it overlaps an E box that has previously been implicated as the binding element for a negative regulator of HIV-1 expression. Through a database search, we identified an additional consensus binding sequence for NF-IL6 in the LTR of many HIV-1 variants and found that over this sequence, purified NF-IL6 can produce an extended footprint that overlaps one of the binding sites for NF-kappa B. A product of the nf-il6 gene activated transcription from several LTR-CAT constructs in transient transfection assays. Thus, NF-IL6 could play a central role in the control of HIV-1 gene expression and this protein might be a key mediator in signaling pathways where HIV-1 is activated by interleukin 6.

Base Sequence↗
PubMed1998

Hematopoietic transcription factor GATA-2 activates transcription from HIV-1 long terminal repeat.

OBJECTIVES: To study the role of the hematopoietic transcription factor GATA-2 in long terminal repeat (LTR)-directed transcriptional activation of HIV-1 in hematopoietic progenitor cells, and to investigate possible GATA-2 binding sites in HIV-1 LTR. DESIGN AND METHODS: Wild-type HIV-1 LTR, or mutants, ligated to a luciferase reporter gene with or without a GATA-2 expression vector, were transfected into COS cells, and standardized luciferase activity was examined. The binding activity of GATA-2 to these sites was examined by electrophoretic mobility shift assay. These wild-type or mutant reporter genes were also transfected into the murine hematopoietic progenitor cells, BAF3, in which GATA-2 was the predominantly expressed transcription factor of the GATA family, to assay LTR-directed transcription in intact hematopoietic machinery. Using a Tat expression plasmid for cotransfection, the influence of Tat protein on GATA-2-induced transactivation was determined. RESULTS: In COS cells, LTR-dependent transactivation was highly enhanced by the coexpression of GATA-2. Experiments with mutant LTR suggested the presence of multiple GATA-2 binding sites, of which the major sites were identified. Cotransfection of Tat with GATA-2 indicated that GATA-2 and Tat synergistically enhanced the transcriptional activity. Transfection experiments in BAF3 cells showed that the disruption of these GATA sites diminished LTR-driven activity to 40% of the wild-type. CONCLUSIONS: GATA-2 may be a key host cell regulator of HIV-1 expression in hematopoietic stem cells. Manipulating this transactivation may represent a valuable approach to controlling virus production in infected hematopoietic progenitors. To elucidate the possible interaction between GATA-2 and Tat protein in vivo might give new insights to the mechanism of impaired hematopoiesis in AIDS patients.

Animals↗
PubMed1991

Variable role of the long terminal repeat Sp1-binding sites in human immunodeficiency virus replication in T lymphocytes.

The long terminal repeat (LTR) of the human immunodeficiency virus (HIV) contains three binding sites for the transcriptional factor Sp1. In order to investigate the role that the Sp1-binding sites play in regulation of HIV replication, we have introduced a deletion of all three Sp1-binding sites into the LTR of an infectious molecular clone of HIV. Viral stocks have been prepared from this mutant virus, designated dl-Sp, and these stocks have been used to study its replicative ability in human T cells. The dl-Sp virus replicated efficiently in MT4 cells and in phytohemagglutinin-stimulated human peripheral blood lymphocytes, but it replicated poorly and with delayed kinetics in A3.01 (CEM) T cells unless those cells had been treated with the cytokine tumor necrosis factor alpha. Gel retardation assays to study the levels of DNA-binding proteins present in these cells showed that NF-kappa B activity could be detected in the nuclei of MT4 cells but not in A3.01 cells unless they had been treated with tumor necrosis factor alpha. Thus, the presence of NF-kappa B activity appeared to be required for efficient replication of an HIV whose LTR Sp1-binding sites had been deleted. This suggests that NF-kappa B can functionally compensate for Sp1 in activating HIV replication. The HIV LTR is therefore similar to the promoter-enhancer units of other viruses in that it is composed of multiple functional elements that may contribute differently to viral replication depending on the levels of DNA-binding proteins present in the target cells.

Animals↗
PubMed1993

Noninvolvement of the long terminal repeat of transposable element 17.6 in insecticide resistance in Drosophila.

Waters and colleagues recently suggested [Waters, L. C., Zelhof, A. C., Shaw, B. J. & Ch'ang, L.-Y. (1992) Proc. Natl. Acad. Sci. USA 89, 4855-4859] that an insertion of a long terminal repeat of transposable element 17.6 into the 3' untranslated region of a P450 gene leads to susceptibility to the insecticide DDT in Drosophila melanogaster. We tested this hypothesis by screening lines from around the world and found that the presence or absence of a long terminal repeat was uncorrelated with resistance in 31 strains of D. melanogaster and Drosophila simulans. Thus we must reject the hypothesis that the insertion of a long terminal repeat leads to DDT susceptibility in Drosophila.

Animals↗
PubMed1983

A unique sequence in murine leukemia virus long terminal repeat functions as a termination signal for transcription in Escherichia coli.

Moloney murine leukemia virus DNA fragments were subcloned into two plasmids downstream of active promoters to detect termination sequences for transcription initiated at these promoters. Two of the viral DNA fragments (2,050 and 595 base pairs) were able to block transcription when inserted in one orientation but not when inserted in the other. These two viral DNA fragments contained long terminal repeat sequences. Deletion of 84 base pairs from the U-3 and R regions in the long terminal repeat removed the transcription termination activity.

Cloning, Molecular↗
PubMed1990

In vivo activation by ultraviolet rays of the human immunodeficiency virus type 1 long terminal repeat.

It has been previously shown in vitro that the human immunodeficiency virus type 1 long terminal repeat (LTR) is activated by ultraviolet irradiation. In order to analyze if a similar effect could occur in vivo, transgenic mice carrying the lacZ gene under the control of the viral LTR were irradiated at 280-300 and 254 nm. These mice spontaneously expressed the transgene in the epidermis and the lens of both adults and embryos. Irradiations caused a significant increase in skin beta-galactosidase activity. This phenomenon might be involved in viral activation and could be of interest in regard to the skin pathology observed during an HIV infection.

Animals↗