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PubMed1996

Targeting of the Sp1 binding sites of HIV-1 long terminal repeat with chromomycin. Disruption of nuclear factor.DNA complexes and inhibition of in vitro transcription.

Sequence selectivity of DNA-binding drugs has recently been reported in a number of studies employing footprinting and gel retardation approaches. In this paper, we studied the biochemical effects of the sequence-selective binding of chromomycin to the long terminal repeat of the human immunodeficiency type I virus. Deoxyribonuclease I (E.C.3.1.21.1) footprinting, arrested polymerase chain reaction, gel retardation and in vitro transcription experiments have demonstrated that chromomycin preferentially interacts with the binding sites of the promoter-specific transcription factor Sp1. Accordingly, interactions between nuclear proteins and Sp1 binding sites are inhibited by chromomycin, and this effect leads to a sharp inhibition of in vitro transcription.

Base Sequence↗
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↗
PubMed1996

Cyclosporin A sensitivity of the HIV-1 long terminal repeat identifies distinct p56lck-dependent pathways activated by CD4 triggering.

The CD4 co-receptor interacts with nonpolymorphic regions of major histocompatibility complex class II molecules on antigen-presenting cells. This interaction results in the mobilization of a number of signaling mediators shared by the T cell receptor (TcR) signaling pathway and thus amplifies TcR-generated signals. We have investigated the outcome of CD4 engagement on the activation of both cellular transcription factors and the HIV-1 long terminal repeat (LTR). We show that CD4 triggering activates different pathways of HIV LTR activation which can be identified by their sensitivity to the immunosuppressant cyclosporin A. The response of the inducible cellular transcription factors involved in HIVLTR activation shows that both nuclear factor (NF)-kappa B and NF-AT mediate a cyclosporin A-sensitive response to CD4, while AP-1 is at least in part responsible for the cyclosporin A-insensitive response. Both pathways can, however, be blocked by a kinase-defective dominant negative p56lck mutant, supporting an essential role for p56lck kinase activity in CD4-dependent signal transduction. A functional analysis of different CD4 epitopes using either anti-CD4 mAb or HIV-1 gp120 reveals a common epitope-specific activation of both the LTR and of the transcription factors NF-kappa B and NF-AT.

Autoantibodies↗
PubMed1996

The effects of 5-fluorouracil and doxorubicin on expression of human immunodeficiency virus type 1 long terminal repeat.

Previous work by many groups has documented induction of the human immunodeficiency virus (HIV) long terminal repeat (LTR) following exposure of cells to ultraviolet light and other DNA damaging agents. Our experiments set out to determine the relative activation or repression of the HIV-LTR in response to two classes of chemotherapeutic agents: Doxorubicin is a DNA damage-inducing agent, and 5-fluorouracil has an antimetabolic mode of action. Using HeLa cells stably transfected with a construct in which HIV-LTR drives expression of the chloramphenicol acetyl transferase reporter gene, we demonstrated an up to ten-fold induction following doxorubicin treatment at 24 h post-treatment. This induction was repressed by treatment with salicylic acid, suggesting a role for prostaglandin/cyclo-oxygenase pathways and/or NF-kappa B in the inductive response. Induction by 5-fluorouracil, in contrast, was more modest (two-fold at most) though it was consistently elevated over controls.

Antibiotics, Antineoplastic↗
PubMed2004

Role of acetylases and deacetylase inhibitors in IRF-1-mediated HIV-1 long terminal repeat transcription.

There is strong evidence that both transcriptional activation and silencing are mediated through the recruitment of enzymes that control reversible protein acetylation: histone acetylase (HAT) and histone deacetylase proteins. Acetylation is also a critical post-translational modification of general and tissue-specific transcription factors. In HIV-1-infected cells, the long terminal repeat (LTR) promoter, once organized into chromatin, is transcriptionally inactive in the absence of stimulation. LTR transcription is regulated by protein acetylation, since treatment with deacetylase inhibitors markedly induces transcriptional activity of the LTR. Besides cellular transcription factors involved in LTR activation, early in infection, and during reactivation from latency, we have previously shown that proteins of the IRF family play an important role. In particular, IRF-1 is able per se to stimulate HIV-1 LTR transcription even in the absence of Tat. IRF-1 is also acetylated and associates with HATs such as p300/CBP and PCAF to form a multiprotein complex that assembles on the promoter of target genes. Here we show that CBP can be recruited by IRF-1 to the HIV-1 LTR promoter even in the absence of Tat and that treatment with deacetylase inhibitors, such as trichostatin A (TSA), increases LTR transactivation in response to both IRF-1 and Tat. These results help to define the architecture of interactions between transcription factors binding HIV-1 LTR and confirm the possibility that deacetylase inhibitors, such as TSA, combined with antiviral therapy may represent a valuable approach to control HIV-1 infection.

Acetylesterase↗
PubMed1992

Transcriptional activity of rel family proteins.

Our studies originally demonstrated that the v-rel oncoprotein repressed gene expression in chicken lymphoid cells, while it activated transcription in rodent fibroblasts. Here we report that the c-rel protein can activate expression of genes linked to kappa B motifs when low levels of endogenous kappa B-binding activity are present. In contrast v-rel, and to a lesser extent c-rel, inhibit NF-kappa B-mediated activation of the human immunodeficiency virus long terminal repeat (HIV LTR) in phorbol ester-stimulated HeLa cells. Competition assays show that v-rel competitively inhibits both NF-kappa B and c-rel-mediated transcriptional activation. Analysis of mutant HIV LTR-chloramphenicol acetyltransferase (CAT) constructs in which all Sp1 or both NF-kappa B elements have been deleted shows that NF-kappa B motifs are required for rel-mediated effects on gene expression. Transforming v-rel mutants compete efficiently with phorbol ester-activated kappa B factors, whereas a transformation-defective mutant of v-rel is impaired in this activity. Taken together, these results strengthen the hypothesis that v-rel functions as a dominant interfering member of rel family proteins. These results also suggest that the ability of v- and c-rel to activate or repress gene expression in specific cells may result from their capacity to compete with endogenous rel family proteins whose expression and/or activity are cell-specific.

Base Sequence↗
PubMed1999

Synthesis, in vitro antiproliferative activity, and DNA-binding properties of hybrid molecules containing pyrrolo[2,1-c][1, 4]benzodiazepine and minor-groove-binding oligopyrrole carriers.

The synthesis, biological activity, and DNA-binding properties of a series of four hybrids prepared by combining polypyrrole minor groove binders and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) 13, related to the naturally occurring anthramycin (3) and DC-81 (4), have been described, and structure-activity relationships have been discussed. These hybrids 22-25 contain from one to four pyrrole units, respectively. To investigate sequence selectivity and stability of drug/DNA complexes, DNase I footprinting and arrested polymerase chain reaction (PCR) were performed on human c-myc oncogene, estrogen receptor gene, and human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) gene sequences. The antiproliferative activity of the hybrids has been tested in vitro on human myeloid leukemia K562 and T-lymphoid Jurkat cell lines and compared to antiproliferative effects of the natural product distamycin A 1, its tetrapyrrole homologue 17, DC 81 (4), and the PBD methyl ester 12. The results obtained demonstrate that the hybrids 22-25 exhibit different DNA-binding activity with respect to both distamycin A 1 and PBD 12. In addition, a direct relationship was found between number of pyrrole rings present in the hybrids 22-25 and stability of drug/DNA complexes. With respect to antiproliferative effects, it was found that the increase in the length of the polypyrrole backbone leads to an increase of in vitro antiproliferative effects, i.e., the hybrid 25 containing the four pyrroles is more active than 22, 23, and 24 both against K562 and Jurkat cell lines.

Antineoplastic Agents↗
PubMed1995

Selective infection of human T-lymphotropic virus type 1 (HTLV-1)-infected cells by chimeric human immunodeficiency viruses containing HTLV-1 tax response elements in the long terminal repeat.

Previous studies have suggested that the human immunodeficiency virus long terminal repeat (HIV LTR) enhancer/promoter sequences contribute to the replication ability of HIV in different T-cell lines; mutation of these sequences can alter HIV tropism. We have utilized site-specific mutagenesis to generate variants of HIV that exhibit specific tropism for human T-lymphotropic virus type 1 (HTLV-1) Tax-expressing CD4+ T cells. The wild-type HIV LTR NF-kappa B and Sp1 sites in an infectious molecular clone of HIV type 1 were replaced with sequences derived from the 21-bp Tax response elements (TRE) from the HTLV-1 LTR to generate TRE-containing chimeric HIVs (TRE-HIVs). The TRE-HIVs exhibit selective replication and cell killing in HTLV-infected human CD4+ T cells, but not in HTLV-negative T cells. Transient transfections suggested that Tax-TRE interactions could account for the observed replication specificity. The TRE-containing HIV LTRs were synergistically activated by the HIV Tat and HTLV-1 Tax transactivators. These results demonstrate that it is possible to specifically target HIV replication and cytotoxicity to HTLV-1+, CD4+ human T cells, on the basis of Tax-TRE interactions, and provide a model for the development of specific, cytotoxic, retroviral gene therapy vectors for HTLV-1-infected cells based on alterations of the LTR transcriptional regulatory elements. They also suggest that HIV Tat can cooperate with heterologous transcriptional activators, such as Tax, which act through upstream binding sites without directly binding to DNA.

Base Sequence↗
PubMed1990

TAR independent activation of the human immunodeficiency virus in phorbol ester stimulated T lymphocytes.

Multiple regulatory elements in the human immunodeficiency virus long terminal repeat (HIV LTR) are required for activation of HIV gene expression. Previous transfection studies of HIV LTR constructs linked to the chloramphenicol acetyltransferase gene indicated that multiple regulatory regions including the enhancer, SP1, TATA and TAR regions were important for HIV gene expression. To characterize these regulatory elements further, mutations in these regions were inserted into both the 5' and 3' HIV LTRs and infectious proviral constructs were assembled. These constructs were transfected into either HeLa cells, Jurkat cells or U937 cells in both the presence and absence of phorbol esters which have previously been demonstrated to activate HIV gene expression. Viral gene expression was assayed by the level of p24 gag protein released from cultures transfected with the proviral constructs. Results in all cell lines indicated that mutations of the SP1, TATA and the TAR loop and stem secondary structure resulted in marked decreases in gene expression while mutations of the enhancer motif or TAR primary sequence resulted in only slight decreases. However, viruses containing mutations in either the TAR loop sequences or stem secondary structure which were very defective for gene expression in untreated Jurkat cells, gave nearly wild-type levels of gene expression in phorbol ester-treated Jurkat cells but not in phorbol ester-treated HeLa or U937 cells. High level gene expression of these TAR mutant constructs in phorbol ester-treated Jurkat cells was eliminated by second site mutations in the enhancer region or by disruption of the tat gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1997

Targeting of the HIV-1 long terminal repeat with chromomycin potentiates the inhibitory effects of a triplex-forming oligonucleotide on Sp1-DNA interactions and in vitro transcription.

We have studied the effects of chromomycin and of a triple-helix-forming oligonucleotide (TFO) that recognizes Sp1 binding sites on protein-DNA interactions and HIV-1 transcription. Molecular interactions between chromomycin, the Sp1 TFO and target DNA sequences were studied by gel retardation, triplex affinity capture using streptavidin-coated magnetic beads and biosensor technology. We also determined whether chromomycin and a TFO recognizing the Sp1 binding sites of the HIV-1 long terminal repeat (LTR) inhibit the activity of restriction enzyme HaeIII, which recognizes a sequence (5'-GGCC-3') located within these Sp1 binding sites. The effects of chromomycin and the TFO on the interaction between nuclear proteins or purified Sp1 and a double-stranded oligonucleotide containing the Sp1 binding sites of the HIV-1 LTR were studied by gel retardation. The effects of both chromomycin and TFO on transcription were studied by using an HIV-1 LTR-directed in vitro transcription system. Our results indicate that low concentrations of chromomycin potentiate the effects of the Sp1 TFO in inhibiting protein-DNA interactions and HIV-1-LTR-directed transcription. In addition, low concentrations of chromomycin do not affect binding of the TFO to target DNA molecules. The results presented here support the hypothesis that both DNA binding drugs and TFOs can be considered as sequence-selective modifiers of DNA-protein interactions, possibly leading to specific alterations of biological functions. In particular, the combined use of chromomycin and TFOs recognizing Sp1 binding sites could be employed in order to abolish the biological functions of promoters (such as the HIV-1 LTR) whose activity is potentiated by interactions with the promoter-specific transcription factor Sp1.

Binding Sites↗
PubMed2001

Selective transduction of HIV-1-infected cells by the combination of HIV and MMLV vectors.

Human immunodeficiency virus 1 (HIV-1)-infected cells are important targets of gene therapy for acquired immune deficiency syndrome. We have developed a novel strategy for targeted gene transfer into HIV-1-infected cells based on 2-step gene transfer. The first step involves the stable introduction of the HIV vector containing the ecotropic Moloney murine leukemia virus (MMLV) receptor gene (EcoRec) into human CD4+ T cells as a molecular switch. Because the HIV-long terminal repeat (HIV-LTR) is Tat inducible, it is expected that EcoRec is expressed only after HIV-1 infection. Northern blot analysis and a retrovirus binding assay confirmed that the HIV-LTR of the integrated vector was silent in transduced cells but strongly transactivated in HIV-1 infection. High levels of EcoRec expression were observed only in HIV-1-infected cells. These cells became highly susceptible to ecotropic MMLV infection and, therefore, in the second step, HIV-1-infected cells were selectively transduced with ecotropic MMLV vectors. More than 70% of HIV-1-infected cells were transduced by this strategy. These findings indicate that this 2-step method can be used for selective and stable gene transfer into HIV-1-infected cells.

Genetic Vectors↗
PubMed2001

Sequence specificity of the human immunodeficiency virus type 2 (hiv-2) long terminal repeat u3 region in vivo allows subtyping of the principal hiv-2 viral subtypes a and b.

Sequences from the nef/LTR overlap region of the human immunodeficiency virus type 2 (HIV-2) genome were amplified from uncultured peripheral blood mononuclear cells (PBMCs) from 40 HIV-2-infected individuals in The Gambia, West Africa. Additional sequences from the plasma of three blood donors were also derived. Analysis of HIV-2 U3 LTR transcription factor elements (PuB-1, p-ets, PuB-2, peri-kappa B, and NF-kappa B sites) indicated a relatively high level of conservation in vivo. The region immediately 3' of the nef termination codon, which exhibits clade-dependent specificity, was targeted by PCR to differentiate HIV-2 subtype A from subtype B infections, the two principal clinical HIV-2 subtypes. All clinical samples analyzed (n = 43) from The Gambia were identified as HIV-2 subtype A by a combination of LTR sequence analysis and subtype-specific amplification of subtypes A and B. Differential PCR amplification of the HIV-2 U3 LTR region represents a rapid means of differentiating subtype A from subtype B infections, the two dominant HIV-2 subtypes that are important in human disease.

Base Sequence↗
PubMed1999

HIV-1 long terminal repeat modulation by glucocorticoids in monocytic and lymphocytic cell lines.

Glucocorticoid gene regulation can be carried out through direct binding of glucocorticoid receptor to glucocorticoid responsive elements (GRE), regulating directly gene transcription and modulating some signaling pathways. The human immunodeficiency virus type 1 (HIV-1) expression can be activated by different immunomodulators through binding of particular nuclear factors to its long terminal repeat (LTR). In order to investigate the effect of glucocorticoids in pathways that activate HIV-1 expression, we transfected promonocyte (U937) and T lymphocyte (CEM-T4) cell lineages with a plasmid containing the chloramphenicol acetyl transferase (CAT) reporter gene under the control of the HIV-1 LTR. In U937 cells, dexamethasone (DEX) downregulates CAT expression induced by either phorbol myristate acetate (PMA), tumor necrosis factor alpha (TNFalpha) or granulocyte/macrophage-colony stimulating factor (GM-CSF). In CEM-T4 cells the CAT activity was slightly upregulated by DEX following the induction by either PMA or TNFalpha. Interestingly, in both cell lines transactivation of this reporter gene by transactivator protein (TAT) was downregulated by DEX. When the CAT gene was under control of HIV-1 enhancer isolated from its LTR background, the CAT activity induced by PMA was not affected by the presence of glucocorticoids. In all experiments, comparable data were obtained when DEX was replaced by hydrocortisone (HC). Our results show that, depending on the cell line, glucocorticoids can differently affect HIV-1 expression, probably by interfering in cellular pathways involved in virus expression. Moreover, the target of this regulation in LTR is probably not the enhancer region itself.

Cell Line↗
PubMed1998

Inhibition of NF-kappaB and HIV-1 long terminal repeat transcriptional activation by inducible nitric oxide synthase 2 activity.

In the human lymphoblastoid T cell line JJhan-5.1, stably transfected with a human immunodeficiency virus-1 long terminal repeat luciferase vector, the level of luciferase activity is dependent on activation of the nuclear factor kappaB (NF-kappaB) transcription factor. Tumor necrosis factor-induced luciferase activity was not modified in JJhan-5.1 cells co-cultivated with murine adenocarcinoma EMT-6 cells but was strongly decreased when nitric oxide (NO) synthase 2 expression was induced in these cells. Two NO synthase inhibitors counteracted this inhibitory effect. Tumor necrosis factor-alpha binding to JJhan-5.1 cells was not modified after incubation with EMT-6 cells. Viability and protein synthesis in JJhan-5.1 cells were also unchanged. Induction of NF-kappaB DNA binding activity was inhibited when EMT-6 cells expressed NO synthase 2 activity. Aminoguanidine, which completely abolished nitrite production, prevented this inhibition. NF-kappaB activation was also strongly inhibited by S-nitrosoglutathione but was marginally affected by N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1, 2-ethylenediamine. Taken together, these results indicated that NO-related species, released by EMT-6 effector cells and probably different from NO itself, inhibited NF-kappaB activation in human lymphoblastoid target cells. Consequently, transcriptional activity of a long terminal repeat-driven luciferase gene construct was markedly diminished.

Animals↗
PubMed1999

No association of HIV type 1 long terminal repeat sequence pattern with long-term nonprogression and in vivo viral replication levels in European subjects.

The HIV-1 long terminal repeat (LTR) promotes and modulates proviral transcription in the infected cell. It has been suggested that truncations and even point mutations in functional sites of the LTR are associated with low viral replication and attenuated pathogenesis in HIV-1-infected long-term nonprogressors (LTNPs). We performed a detailed analysis of LTR sequences from proviral DNA of 21 Italian and Swedish, well-characterized LTNPs and of 15 progressor patients. No truncation was found and no correlation was identified between specific LTR mutations and disease progression. We also failed to find a significant correlation between phylogenetic distance and clinical status. Although HIV-1 LTR interpatient heterogeneity among LTNPs and subjects with HIV-1 RNA levels <500 copies/ml tended to be lower, no sequence mutation was correlated with in vivo viral loads. Our results suggest that HIV-1 LTR defects are rare among Italian and Swedish LTNPs.

Adolescent↗
PubMed1996

Binding of human immunodeficiency virus type 1 to CD4 induces association of Lck and Raf-1 and activates Raf-1 by a Ras-independent pathway.

We have analyzed CD4-mediated signaling during the early stages of human immunodeficiency virus type 1 (HIV-1) infection. Binding of purified HIV-1 virions or recombinant HIV-1 glycoprotein gp120 to CD4 receptors resulted in association and tyrosine phosphorylation and activation of tyrosine kinase Lck and serine/threonine kinase Raf-1. The association between Lck and Raf-1 was mediated by stimulation of the CD4 receptors, since it was abolished by preincubation of the virus with soluble CD4 and was not detected in CD4-negative A201 T cells. However, the Lck-Raf-1 association was restored in A201 cells permanently transfected with human CD4 cDNA and stimulated with anti-CD4 antibodies. In addition, a catalytically active Lck was required for the association of Lck and Raf-1. Surprisingly, the CD4-mediated signaling, induced by the HIV-1 binding, did not result in stimulation of the Ras GTP-binding activity or its association with Raf-1, indicating that the signaling pathway generated by the HIV-1 binding is not identical to the classical Ras/Raf-1 pathway. Furthermore, overexpression of activated Raf-1 in Jurkat T cells stimulated the HIV long terminal repeat promoter activity and significantly enhanced HIV-1 replication. This suggests that the Lck-Raf-1 pathway, rapidly stimulated by the binding of HIV-1 or gp120 to CD4 receptors, may play an essential role in the transcriptional activation of the integrated HIV-1 provirus as well as in its pathogenicity.

Antibodies↗
PubMed1995

CD8+ T cells suppress human immunodeficiency virus replication by inhibiting viral transcription.

CD8+ cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in cultured CD4+ cells by a noncytolytic mechanism that involves a secreted CD8(+)-cell antiviral factor (CAF). The results of this study suggest that CD8+ cells, as well as CAF, arrest HIV replication at the level of viral transcription. Culturing naturally infected CD4+ cells actively producing HIV with autologous CD8+ cells or a 50% dilution of culture fluids from these cells results in a > 80% reduction in the number of cells expressing HIV antigens and RNA. This effect was observed within 2 days after exposure to CD8+ cells but required 6 days in the presence of CAF-containing culture fluids to reach the same extent of HIV suppression. Northern blot analysis of CD4+ cell extracts revealed that all viral RNA species (unspliced and single and double spliced) were reduced in quantity to a similar extent. CAF-containing culture fluids also had a direct inhibitory effect on HIV long terminal repeat (LTR)-driven transcription in HIV-infected 1G5 cells carrying an LTR-luciferase construct. Suppression of basal levels of LTR-driven transcription was not detected. Thus, the results suggest that the noncytolytic CD8+ cell antiviral activity observed in HIV infection exerts its effects, at least in part, by specifically interrupting HIV transcription. These findings could help in developing therapies for HIV infection.

Antiviral Agents↗