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PubMed1992

Hexamethylene bisacetamide stimulates the expression of human immunodeficiency virus long terminal repeat sequences in rat and human fibroblasts.

We have employed a recombinant plasmid, pBHIV1, carrying the long terminal repeat (LTR) sequence of the human immunodeficiency virus-1 (HIV-1) linked to the reporter chloramphenicol acetyl transferase (CAT) gene and to the aminoglycoside phosphotransferase (aph) gene as a selectable marker. We have introduced pBHIV1 in rat 208F and human MRCSV40TGR fibroblasts and obtained stable geneticin resistant RFBHIV1-1 and SVTGHIV1-1 cells, respectively. Both transfectant cells express CAT activity from the HIV LTR promoter. The response to anti-neoplastic drug hexamethylene bisacetamide (HMBA) was studied on the LTR regulated CAT activity in both cell lines. It was found that HMBA at 5mM concentration stimulates the expression of CAT from the HIV LTR in rat and human cells by 28- and 1.9-fold, respectively.

Acetamides↗
PubMed1989

Multiple transcriptional regulatory domains in the human immunodeficiency virus type 1 long terminal repeat are involved in basal and E1A/E1B-induced promoter activity.

The human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR) is the site of activation of the HIV tat protein. However, additional transactivators, such as the adenovirus E1A and herpesvirus ICPO proteins, have also been shown to be capable of activating the HIV LTR. Analysis of adenovirus mutants indicated that complete transactivation of the HIV LTR was dependent on both the E1A and E1B proteins. To determine which regions of the HIV LTR were important for complete E1A/E1B activation, a variety of oligonucleotide-directed mutations in HIV transcriptional regulatory domains were assayed both in vivo and in vitro. S1 nuclease analysis of RNA prepared after transfection of these HIV constructs into HeLa cells infected with wild-type adenovirus indicated that the enhancer, SP1, TATA, and a portion of the transactivation-responsive element were each required for complete E1A/E1B-mediated activation of the HIV LTR. These same promoter elements were required for both basal and E1A/E1B-induced levels of transcription in in vitro transcription reactions performed with cellular extracts prepared from cells infected with dl434, an E1A/E1B deletion mutant, or wild-type adenovirus. No mutations were found that reduced only E1A/E1B-induced expression without proportionally reducing basal levels of transcription, suggesting that E1A/E1B-mediated induction of the HIV LTR requires multiple promoter elements which are also required for basal transcriptional levels. Unlike activation by the tat protein, there was not a rigid dependence on maintenance of the transactivation-responsive stem base pairing for E1A/E1B-mediated activation either in vivo or in vitro, indicating that activation occurs by a mechanism distinct from that of tat induction.

Adenovirus Early Proteins↗
PubMed1989

The NF-kappa B binding sites in the human immunodeficiency virus type 1 long terminal repeat are not required for virus infectivity.

Mutations were introduced into the regulatory sequences in the long terminal repeat of an infectious molecular clone of the human immunodeficiency virus. Viruses in which the NF-kappa B binding sites were deleted or ones in which one or two Sp1 binding sites were mutated still replicated efficiently in human T lymphocytes. A deletion of the two NF-kappa B sites plus the three Sp1 sites or a mutation of the tat-responsive region rendered the virus replication incompetent. Thus, the NF-kappa B sequences are not required for human immunodeficiency virus infectivity; however, a tat-responsive region is essential.

Base Sequence↗
PubMed1995

Role of the TATA box in transcription of the mouse mammary tumor virus long terminal repeat.

An in vitro transcription system from mammary cells was established to study transcription of the long terminal repeat (LTR) of the mouse mammary tumor virus (MMTV). Experiments with progressive 5'-deletion constructs of the MMTV LTR revealed that a 19-base pair (bp) region from -41 to -23 bp, encompassing the TATA box and flanking DNA sequence, was as transcriptionally active as larger promoter constructs, both in nuclear extracts from human mammary cell lines (T47D and MCF7) and a nonmammary cell line (HeLa). The cell-free system was capable of supporting transcriptional induction by factors binding upstream of the TATA box, however, since purified glucocorticoid receptor-induced transcription in larger promoter constructs encompassing the MMTV hormone-responsive elements. Transcription from two other promoters, the adenovirus major late promoter and the human immunodeficiency virus LTR, also revealed a significant transcriptional contribution of upstream elements. The 19-bp TATA region from the MMTV LTR was shown to have considerably more activity in this transcription system than comparable TATA regions from other promoters. Sequences critical to the MMTV TATA region were evaluated by single base pair mutagenesis and found to comprise a consensus TATA box sequence, TATAAAA, as well as a single A just upstream of the TATAAAA sequence. Thus, the high level of basal transcription observed with the TATA region from MMTV is due to a perfect consensus TATA box sequence and a single base immediately 5' adjacent. It is likely that the high basal rate of transcription observed with this TATA box region on histone-free templates represents an inappropriate level of basal expression and that a complete evaluation of transactivation mechanisms in this system will require the recapitulation in vitro of the chromatin-mediated repressive state that exists in vivo.

Base Sequence↗
PubMed1991

Identification of a hypervariable region in the long terminal repeat of equine infectious anemia virus.

An avirulent, field-derived isolate of equine infectious anemia virus (EIAV), designated MA-1, was molecularly cloned, and the complete nucleotide sequence was determined for the 3' half of the viral genome. Comparisons between MA-1 and the prototype Wyoming strain of EIAV identified a 66-nucleotide stretch between CAAT (-91) and TATAA (-25) in the U3 region of the long terminal repeat, where sequence divergence was as high as 39.3%. The polymerase chain reaction was used to amplify and clone long terminal repeat sequences from Th-1, the in vivo parental stock of MA-1. Results indicated that the nucleotide sequences of MA-1 and Th-1 clones were less variable than was observed between MA-1 and Wyoming. However, MA-1 and Th-1 markedly differed in the types of enhancer sequences located in the hypervariable region. These results suggest that variation in lentivirus regulatory sequences may be important in EIAV host cell tropism and pathogenesis.

Amino Acid Sequence↗
PubMed1997

COUP-TF and Sp1 interact and cooperate in the transcriptional activation of the human immunodeficiency virus type 1 long terminal repeat in human microglial cells.

We have recently reported that chicken ovalbumin upstream promoter transcription factor (COUP-TF) activates human immunodeficiency virus type 1 (HIV-1) gene transcription in glial and neuronal cells. Here, we have examined the role of COUP-TF in microglial cells, the major target cells for HIV-1 infection in brain. We show that COUP-TF activates gene expression from both the lymphotropic LAI and the macrophage-tropic JR-FL HIV-1 strains. Although COUP-TF binds to the -352/-320 nuclear receptor responsive element of the long terminal repeat, it functions as a transcriptional activator by acting on the -68/+29 minimal promoter. This region is a direct target of transcription factors Sp1 and Sp3. We report the discovery and features of a physical and functional interplay between COUP-TF and Sp1. Our cotransfection experiments provide evidence for a functional synergism between Sp1 and COUP-TF leading to enhanced transcriptional activity of the HIV-1 long terminal repeat through the Sp1 element. In contrast, Sp3 functions as a repressor of Sp1- or COUP-TF-induced activation. We further demonstrate that COUP-TF and Sp1 are capable of physically interacting, via the DNA-binding domain of COUP-TF, in vitro and in the cell. These findings reveal how the novel interplay of Sp1 and COUP-TF families of transcription factors regulate HIV-1 gene expression.

COUP Transcription Factor I↗
PubMed1990

Substitution of the U3 long terminal repeat region of the neurotropic Cas-Br-E retrovirus affects its disease-inducing potential.

The Cas-Br-E and ts-Mo BA-1 murine leukemia viruses (MuLV) induce a spongiform neurodegenerative disease with different clinical manifestations, namely, either hind limb paralysis (Cas-Br-E) or tremors, spasticity, and hind limb weakness (ts-Mo Ba-1). We constructed the chimeric NEBA-1 MuLV by replacing the long terminal repeat of Cas-Br-E MuLV with that of ts-Mo BA-1 MuLV. In SWR/J or CFW/D mice, NEBA-1 MuLV induced an ataxic neurological disease characterized by clinical signs different from those induced by both parents. Although NEBA-1 MuLV did not induce lesions in novel brain areas, the spongiform lesions were more severe in deep cerebellar nuclei and in the spinal cord than those found in paralyzed mice inoculated with Cas-Br-E MuLV. By in situ hybridization, we found that the distribution of the spongiform lesions closely correlated with the distribution of the infected central nervous system cells. In the spinal cord, a close correlation was found between the number of infected cells and the severity of the spongiform degeneration. Sequencing of the substituted ts-BA-1 MuLV fragment and comparison with homologous sequences of Cas-Br-E and Moloney MuLV showed differences mainly in the U3 tandem direct repeats. Our results show that a few modifications within the U3 long terminal repeat allow the virus to cause more severe lesions in some central nervous system regions and that the severity of the spongiform degeneration correlates with the level of viral replication.

Animals↗
PubMed1996

A fourth Sp1 site in the human immunodeficiency virus type 1 long terminal repeat is essential for negative-sense transcription.

We report the discovery of a fourth Sp1 binding site at the 5' end of the U3 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (HIV-1 Sp1 IV), localized to HXB2 nucleotides -433 to -441. This site is shown to bind Sp1 protein specifically in electrophoretic mobility shift assays. Sp1 protein appears to bind to HIV-1 Spl IV with 5 to 10 times lower affinity than to a consensus Sp1 site. Mutation of HIV-1 Sp1 IV in an HXB2-derived long terminal repeat-chloramphenicol acetyltransferase reporter construct gave no significant change in positive-sense transcription but abolished both basal and phorbol myristate acetate-activated negative-sense transcription. Taken together, the results further define the HIV-1 negative-sense promoter as an Sp1-dependent, phorbol myristate acetate-responsive, and Tat-inhibited promoter initiating at HXB2 nucleotide -450.

Antisense Elements (Genetics)↗
PubMed1984

Elevated expression of an endogenous retroviral long terminal repeat in a mouse colon tumor.

We have previously identified a number of cloned poly(A)+ RNA sequences in a cDNA library, each of whose expression is markedly altered in a dimethylhydrazine-induced mouse colon tumor as compared to the normal colon. Transcripts which hybridize to one of the cloned cDNAs, pMCT-1, have now been shown to be elevated in expression in three mouse leukemia cell lines as well, and induction of differentiation in one of these lines (DS-19) is associated with a substantial decrease in expression of these transcripts. By hybridization and sequence analysis, pMCT-1 has been found to contain a region homologous to a long terminal repeat of an intracisternal A particle sequence. Intracisternal A sequences are endogenous, retroviral genomes repeated several thousandfold and dispersed in the mouse genome. The data show that the transcripts which are homologous to this long terminal repeat are highly heterogeneous, suggesting that many intracisternal type A particle transcriptional units may be expressed in the colon tumor and leukemia cell lines.

Animals↗
PubMed2004

Identification of mutations in proviral long terminal repeats of HIV type 1-infected subjects naive to drug therapy.

Human immunodeficiency virus type 1 (HIV-1) proviral DNA sequences in the 5' long terminal repeat (LTR) were examined among 28 drug-naive individuals. Twenty-four subjects had highly conserved LTR sequences, however, more significant changes were observed in the remaining four LTR sequences. These included a 9-bp deletion preceding the NF kappa B elements and a duplication of the RBF-2 motif. A higher overall frequency of mutations within the LTR occurred within NFAT-1 and Sp-1 sequences. Importantly, a novel 16-bp deletion was found in the distal NFAT-1 site.

Base Sequence↗
PubMed2006

5' long terminal repeat (LTR)-selective methylation of latently infected HIV-1 provirus that is demethylated by reactivation signals.

We previously described selective hypermethylation of the 5'-long terminal repeat (LTR) of HTLV-1 provirus in vivo and in vitro. This prompted us to analyze CpG methylation of the two LTRs of the HIV provirus in chronically infected cell lines. The results demonstrate selective hypermethylation of the 5' LTR of the HIV provirus in ACH-2 cells. Moreover, induction of viral gene expression by TNF-alpha resulted in demethylation of the 5'-LTR. These results suggest that selective epigenetic modification of the 5'LTR of the HIV-1 provirus may be an important mechanism by which proviral activity is suppressed.

Base Sequence↗
PubMed1992

Transcellular activation of the human immunodeficiency virus type 1 long terminal repeat in T lymphocytes requires CD4-gp120 binding.

Cells expressing human immunodeficiency virus type 1 (HIV-1) tat can transactivate the HIV-1 long terminal repeat (LTR) in cocultured T lymphocytes. In this report, we describe the molecular requirements for transcellular activation of the LTR in Jurkat cells. An analysis with deletion mutants and blocking antibodies demonstrated a requirement for env expression in addition to tat expression for transcellular activation to occur. The results suggest that the transient association of CD4 and gp120 in cocultured cells is required for tat-mediated transcellular activation. The events that follow CD4-gp120 binding in transactivation, however, do not require the gp120-neutralizing domain, in contrast to HIV-mediated fusion and infection. The consequences of this interaction on cellular function are currently under investigation.

CD4 Antigens↗
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↗
PubMed1999

Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription.

Transcriptional activation of the HIV type 1 (HIV-1) long terminal repeat (LTR) promoter element by the viral Tat protein is an essential step in the HIV-1 life cycle. Tat function is mediated by the TAR RNA target element encoded within the LTR and is known to require the recruitment of a complex consisting of Tat and the cyclin T1 (CycT1) component of positive transcription elongation factor b (P-TEFb) to TAR. Here, we demonstrate that both TAR and Tat become entirely dispensable for activation of the HIV-1 LTR promoter when CycT1/P-TEFb is artificially recruited to a heterologous promoter proximal RNA target. The level of activation observed was indistinguishable from the level induced by Tat and was neither inhibited nor increased when Tat was expressed in trans. Activation by artificially recruited CycT1 depended on the ability to bind the CDK9 component of P-TEFb. In contrast, although binding to both Tat and TAR was essential for the ability of CycT1 to act as a Tat cofactor, these interactions became dispensable when CycT1 was directly recruited to the LTR. Importantly, activation of the LTR both by Tat and by directly recruited CycT1 was found to be at the level of transcription elongation. Together, these data demonstrate that recruitment of CycT1/P-TEFb to the HIV-1 LTR is fully sufficient to activate this promoter element and imply that the sole role of the Tat/TAR axis in viral transcription is to permit the recruitment of CycT1/P-TEFb.

Animals↗
PubMed1996

Activation of the transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat by autologous and heterologous cell-to-cell contact.

Recent studies have demonstrated that the activation of HIV-1 provirus and of the Long Terminal Repeat of HIV-1 (HIV-1-LTR) may occur upon cell-to-cell contact between peripheral blood lymphocytes (PBLs) and infected or transfected (HT29) human colonic carcinoma cells. Using transient or stable transfections, we ascertained that the HIV-1 LTR was up-regulated by cell-to-cell contact in various cell lines. The degree of cell-to-cell contact responsiveness was cell type dependent. In contrast, in transient transfection, the HIV-1-LTR was strongly induced by Tat expression in all cell types tested. This indicates that there are differences in the induction mechanism for these two stimuli, even though Tat protein has been previously reported to induce cell adhesion. Except for the PBLs/transfected cells interactions, the results also demonstrate that the cell-to-cell contact-induced HIV-1-LTR activation was highest after contact between autologous as compared to heterologous cells. Previous experiments have shown that, in HT29 cells, cell-to-cell contact activation of the HIV-1-LTR involved the NF-kappa B tandem binding site and activates DNA binding of the nuclear factor NF-kappa B. In this work, we show that in a stably transfected cell line, the principal enhancer element was also involved in the HIV-1-LTR cell-to-cell contact activation. On the other hand, in the HT29 cell line, the NF-kappa B binding appeared to involve the RGD motif of the cell-binding domain, which indicates that a post-integrin receptor event could be implicated.

Cell Communication↗
PubMed1991

Linker-scanning mutational analysis of the transcriptional activity of the human immunodeficiency virus type 1 long terminal repeat.

We have compared the relative importance of transcription regulatory regions in the U3 and R regions of the human immunodeficiency virus type 1 long terminal repeat (LTR) by using linker-scanning mutational analysis. Twenty-six mutant LTR-chloramphenicol acetyltransferase (CAT) transient expression plasmids were prepared in which consecutive 18-bp regions of wild-type LTR were replaced with an NdeI-XhoI-SalI (NXS) polylinker. The mutant LTR-CAT plasmids were transfected into unstimulated Jurkat cells, Jurkat cells stimulated with phytohemagglutinin and tetradecanoylphorbol acetate, and Jurkat cells which constitutively express the human immunodeficiency virus type 1 trans-activator protein, Tat. Transcriptional activity was measured by analysis of CAT activity. The activities of these mutants identified one major and several minor transcription control elements in addition to previously identified elements. In addition, this fine-structure analysis identified differences in utilization of regulatory regions between unstimulated, stimulated, and Tat-expressing Jurkat cells. A significant regulatory region was indicated by linker-scanning mutations between nucleotides -183 and -130 (relative to the transcription start site, +1). These mutations caused marked decreases in activity of the LTR in unstimulated and especially in stimulated Jurkat cells but had no effect in Tat-expressing Jurkat cells. DNA mobility shift studies comparing probes of wild-type and mutant sequences in the -183 to -130 region indicated that alterations in specific DNA binding correspond to the altered transcriptional activity of the mutants. The effects of mutations in several regulatory regions, in addition to the -183 to -130 region described above, differ between Tat-expressing and -nonexpressing Jurkat cells. For example, the NF-kB sites are necessary for transcription in both Tat-expressing and -nonexpressing cells. However, Tat-expressing Jurkat cells primarily require only the 3'-proximal site, while both stimulated and unstimulated Jurkat cells appear to require both sites. Mutants downstream of the TATA element cause a more significant decrease in activity in Tat-expressing Jurkat cells than in the others. Finally, several mutations in the 5' half of the LTR (-453 to -184) show modest increases in transcription (1.5-fold or less) in unstimulated Jurkat cells only, suggesting possible negative regulatory sites. In summary, our studies have identified a control region (-183 to -130) upstream of the NF-kB sites and have more precisely defined significant differences in the utilization of regulatory regions between unstimulated, stimulated, and Tat-expressing Jurkat cells.

Base Sequence↗
PubMed1988

Structural and functional analysis of long terminal repeats of Suncus murinus mammary tumor virus.

A 2.7-kilobase (kb) cDNA sequence complementary to Suncus murinus mammary tumor virus (Sm-MTV) genomic RNA [corrected] was prepared using purified virions produced by the Sm-MT cell line, which had been established from a spontaneous mammary tumor of S. murinus. It was found, by using this cDNA in Southern hybridization experiments, that Sm-MTV was endogenous to this animal and that some 50 copies of endogenous provirus were present per haploid cellular genome. In addition, a proviral Sm-MTV DNA sequence, 9.4 kb long (Sm-P-MTV10), was cloned from a Sm-MT cell genomic library, and its long terminal repeat was found to be 720 base pairs (bp) long, with the U3.R and U5 regions 574 and 146 bp long, respectively. The boundary between U3 and R was not determined with certainty, though in the cDNA, the U3 and R regions were 462 and 105 bp long, respectively. The overall homology between the U3.R regions in the cDNA and Sm-P-MTV was 75%. These two DNAs differed in such transcription regulatory signals as CCAAT and TATAA, the first being missing from the cDNA. Nevertheless, chloramphenicol acetyltransferase assays showed that the long terminal repeats of the cDNA and the Sm-P-MTV were transcriptionally active but not steroid hormone responsive. Like Mason-Pfizer monkey virus, Sm-MTV used tRNA(1,2Lys) as a primer for reverse transcription. In addition, the immunosuppressive peptide sequence common to many retroviruses was found in the env region of Sm-MTV. In these two points, Sm-MTV differed from mouse MTV.

Animals↗
PubMed2002

A retroviral long terminal repeat adjacent to the HLA DQB1 gene (DQ-LTR13) modifies Type I diabetes susceptibility on high risk DQ haplotypes.

AIMS/HYPOTHESIS: HLA-DQ genes, located in the human leukocyte antigen region on chromosome 6 p, are the main inherited factors predisposing to Type I (insulin-dependent) diabetes mellitus. Endogenous retroviral long-terminal repeats are integrated at several sites within this region, one of which is known to enhance susceptibility for Type I diabetes. We examined another LTR within the HLA-region as an additional genetic risk marker. METHODS: We investigated the segregation of one long-terminal repeat (DQ-LTR13), located 1.3 kb upstream of HLA DQB1 with different HLA-DQ haplotypes, and its transmission to patients. A total of 284 Caucasian families (203 German and 81 Belgian) with at least one diabetic offspring were genotyped for DQA1, DQB1 and DQ-LTR13. RESULTS: DQ8/LTR13(+) was preferentially transmitted (139 transmitted vs 28 not transmitted; P(TDT) = 1.67 x 10(-14)) whereas no deviation from expected transmission frequencies was observed for DQ8/LTR13(-) (20 transmitted vs 17 not transmitted; P(TDT) = 1.00). DQ8/LTR13(+) alleles conferred a significantly higher risk for Type I diabetes than DQ8/LTR13(-) alleles (p chi(2) = 2.58 x 10(-14)). This difference remained significant even after DRB1 subtyping (p chi(2) = 0.02). Also, there was a significant difference when comparing the transmission of DQ2/LTR13(+) and DQ2/LTR13(-) alleles (p chi(2) = 0.01), the latter conferring an increased risk. The transmission of DQ-LTR13(+) haplotypes did not show any differences regarding paternal, maternal or gender-related stratification. However, DQ8/LTR13(-) was significantly more often transmitted from mothers (p chi(2) = 0.01) and to female patients (p chi(2) = 0.04). CONCLUSION/INTERPRETATION: We conclude that DQ-LTR13 marks additional genetic risk for Type I diabetes on predisposing DRB1(*)0401- DQ8 and DQ2 haplotypes and will help to further define susceptibility in this gene region.

Base Sequence↗