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Results for “HIV Long Terminal Repeat”

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PubMed2001

Modulation of cellular and viral gene expression by the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is the likely etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Common to these malignancies is that tumor cells are latently infected with KSHV. Viral gene expression is limited to a few genes, one of which is the latency-associated nuclear antigen (LANA), the product of ORF73. Examination of the primary sequence of LANA reveals some structural features reminiscent of transcription factors, leading us to hypothesize that LANA may regulate viral and cellular transcription during latency. In reporter gene-based transient transfection assays, we found that LANA can have either positive or negative effects on gene expression. While expression of a reporter gene from several synthetic promoters was increased in the presence of LANA, expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR)-and from NF-kappaB-dependent reporter genes-was reduced by LANA expression. In addition, the promoter of KSHV ORF73 itself is activated up to 5.5-fold by LANA. This autoregulation may be important in tumorigenesis, because two other genes (v-cyclin and v-FLIP) with likely roles in cell growth and survival are also controlled by this element. To identify cellular genes influenced by LANA, we employed cDNA array-based expression profiling. Six known genes (and nine expressed sequence tags) were found to be upregulated in LANA-expressing cell lines. One of these, Staf-50, is known to inhibit expression from the HIV LTR; most of the other known genes are interferon inducible, although the interferon genes themselves were not induced by LANA. These data demonstrate that LANA expression has effects on cellular and viral gene expression. We suggest that, whether direct or indirect in origin, these effects may play important roles in the pathobiology of KSHV infection.

Animals↗

PubMed1999

Thymocyte-thymic epithelial cell interaction leads to high-level replication of human immunodeficiency virus exclusively in mature CD4(+) CD8(-) CD3(+) thymocytes: a critical role for tumor necrosis factor and interleukin-7.

This work aims at identifying the thymocyte subpopulation able to support human immunodeficiency virus (HIV) replication under the biological stimuli of the thymic microenvironment. In this report we demonstrate that interaction with thymic epithelial cells (TEC) induces a high-level replication of the T-tropic primary isolate HIV-1(B-LAIp) exclusively in the mature CD4(+) CD8(-) CD3(+) thymocytes. Tumor necrosis factor (TNF) and interleukin-7 (IL-7), secreted during this interaction, are critical cytokines for HIV long terminal repeat transactivation through NF-kappaB-dependent activation. TNF is the major inducer of NF-kappaB and particularly of the p50-p65 complex, whereas IL-7 acts as a cofactor by sustaining the expression of the p75 TNF receptor. The requirement for TNF is further confirmed by the observation that the inability of the intermediate CD4(+) CD8(-) CD3(-) thymocytes to replicate the virus is associated with a defect in TNF production during their interaction with TEC and correlates with the absence of nuclear NF-kappaB activity in these freshly isolated thymocytes. Addition of exogenous TNF to the intermediate thymocyte cultures induces NF-kappaB activity and is sufficient to promote HIV replication in the cocultures with TEC. The other major subpopulation expressing the CD4 receptor, namely, the double-positive (DP) CD4(+) CD8(+) CD3(+/-) thymocytes, despite the entry of the virus, do not produce a significant level of virus, presumably because they are unresponsive to TNF and IL-7. Together, these data suggest that in vivo, despite an efficient entry of the virus in all the CD4(+) subpopulations, a high viral load may be generated exclusively within the mature CD4(+) CD8(-) CD3(+) subset of thymocytes. However, under conditions of inflammatory response after infection, TNF might also be present in the intermediate thymocyte compartment, leading to efficient HIV replication in these cells.

Antigens, CD↗

PubMed1992

Contribution of the TATA motif to Tat-mediated transcriptional activation of human immunodeficiency virus gene expression.

Tat-mediated transcriptional activation of human immunodeficiency virus (HIV) gene expression requires the presence of the cis-acting Tat-responsive element, TAR, and a functional enhancer-promoter element. The ability of Tat to function with heterologous enhancer sequences led us to examine the role of the minimal basal promoter for trans activation. Substitution of HIV TATA sequences (nucleotides -20 to -35) with TATA elements derived from other promoters had little effect on the basal level of transcription or the ability to activate the HIV long terminal repeat upon stimulation through upstream activation sequences. In contrast, minimal alterations within the TATA motif had a profound effect on trans activation, as demonstrated by the 3- to 10-fold reduction in activation following expression of Tat. Our findings suggest that minor changes in the TATA motif affect the composition of the initiation-elongation complex and that the composition of this complex is critical for Tat-dependent activation of gene expression.

Animals↗

PubMed1994

High activity and stability of hammerhead ribozymes containing 2'-modified pyrimidine nucleosides and phosphorothioates.

The influence of chemical modifications on the catalytic activity and stability of a hammerhead ribozyme directed against the long terminal repeat RNA of the human immunodeficiency virus 1 was examined. Previous studies had shown that substitution of all pyrimidine nucleosides by their 2'-fluoro analogs led to an 8-fold decrease in catalytic efficiency in the cleavage reaction compared to the unmodified ribozyme (Heidenreich, O., and Eckstein, F. (1992) J. Biol. Chem. 267, 1904-1909). It is shown here that replacement of the 2'-fluoro-2'-deoxyuridines in the conserved region of this ribozyme, positions 4 and 7, by 2'-amino-2'-deoxyuridines fully restores catalytic activity of the ribozyme. Ribozymes containing these 2'-modifications show an increased stability against RNases present in fetal calf serum and in cell culture supernatant. The stability is increased further by the incorporation of four terminal phosphorothioates as protection against 3'-exonucleases, the degree of which depends on the secondary structure of the ribozyme. Such ribozymes are stable in undiluted fetal calf serum for at least 24 h. The results clearly demonstrate the potential to design stable ribozymes without any loss of catalytic activity.

Animals↗

PubMed1992

Hammerhead ribozyme-mediated cleavage of the long terminal repeat RNA of human immunodeficiency virus type 1.

Three ribozymes targeted against different sites of the long terminal repeat RNA (LTR RNA) of human immunodeficiency virus type 1 cleaved a LTR RNA transcript 1,000 x less efficiently than corresponding short synthetic oligoribonucleotide substrates. Varying the stem lengths of the ribozyme resulted in changes of the catalytic efficiency. Almost no cleavage was observed for a ribozyme forming only 10 base pairs instead of 14 with the LTR RNA. Increasing the base pairs to 16 or elongation of the stem formed within the ribozyme revealed only small changes in kcat/Km. The influence of chemical modifications within the ribozyme on the cleavage of the LTR RNA was also examined. 2'-Fluorocytidine substitutions as well as four terminal phosphorothioate internucleotidic linkages influenced the catalytic efficiency of ribozymes only negligibly. However, substitution of uridine by 2'-fluorouridine resulted in a 5-fold decrease of kcat/Km. A ribozyme containing all these modifications revealed only a 7-fold lower catalytic efficiency but a markedly increased stability in cell culture supernatant. These results demonstrate that it is possible to increase the stability of ribozymes toward nucleases without a serious loss in catalytic efficiency.

Base Sequence↗

PubMed1988

The Fos complex and Fos-related antigens recognize sequence elements that contain AP-1 binding sites.

The Fos protein complex and several Fos-related antigens bind directly or indirectly to a common sequence element that is similar to the consensus binding site for HeLa cell activator protein 1 (AP-1). This element is present in a negative regulatory sequence in the differentiation-sensitive adipocyte gene, aP2; in a transcriptional enhancer for the Gibbon ape leukemia virus; and in a region of the human immunodeficiency virus (HIV) long terminal repeat partially characterized as a negative regulatory element. The protein level and binding activity of Fos and Fos-related antigens increase rapidly after calcium ionophore treatment of a CD4+ human lymphoblast cell line, H9. These data suggest that several proteins may associate with the AP-1 binding site. Moreover, temporally regulated control of the level of each protein could represent a mechanism for modulation of these putative mediators of gene expression.

Base Sequence↗

PubMed1993

Juxtaposition between activation and basic domains of human immunodeficiency virus type 1 Tat is required for optimal interactions between Tat and TAR.

trans activation of the human immunodeficiency virus type 1 long terminal repeat requires that the viral trans activator Tat interact with the trans-acting responsive region (TAR) RNA. Although the N-terminal 47 amino acids represent an independent activation domain that functions via heterologous nucleic acid-binding proteins, sequences of Tat that are required for interactions between Tat and TAR in cells have not been defined. Although in vitro binding studies suggested that the nine basic amino acids from positions 48 to 57 in Tat bind efficiently to the 5' bulge in the TAR RNA stem-loop, by creating several mutants of Tat and new hybrid proteins between Tat and the coat protein of bacteriophage R17, we determined that this arginine-rich domain is not sufficient for interactions between Tat and TAR in vivo. Rather, the activation domain is also required and must be juxtaposed to the basic domain. Thus, in vitro TAR RNA binding does not translate to function in vivo, which suggests that other proteins are important for specific and productive interactions between Tat and TAR.

Amino Acid Sequence↗

PubMed1998

The single-stranded DNA binding protein, Pur-alpha, binds HIV-1 TAR RNA and activates HIV-1 transcription.

Previous studies indicate that the bulge and loop domains of TAR, the HIV-1 RNA regulatory element, bind viral and cellular factors that are critical for efficient transcription of the HIV-1 genome. In this report, we demonstrate that the cellular protein, Pur-alpha, a previously characterized sequence specific, single-stranded DNA binding protein, binds to HIV-1 TAR RNA in a specific manner as demonstrated by competition analysis. Pur-alpha binds to the greatest extent to wild-type TAR RNA, and it appears the primary sequence, as well as the secondary structure and its overall stability contribute to this binding. Results from gel shift analysis using mutant Pur-alpha proteins indicate that amino acids 55-85, which contain the first of three basic aromatic repeats, are important for its binding to TAR RNA. Overexpression of Pur-alpha in a glial cell line increased transcription of HIV-1 LTR by a TAR dependent mechanism. The potential contribution by Pur-alpha to HIV-1 expression in relation to basal transcription by cellular factors is discussed.

Base Sequence↗

PubMed1993

Functional analysis of interactions between Tat and the trans-activation response element of human immunodeficiency virus type 1 in cells.

Transcriptional trans-activation of the human immunodeficiency virus type 1 long terminal repeat requires that the virally encoded Tat effector interacts with its target trans-activation response element (TAR) RNA stem-loop. Although the arginine-rich region of Tat from amino acids 49 to 59 is sufficient to bind to TAR RNA in vitro, the RNA-binding domain of Tat has not been defined in vivo. Human immunodeficiency virus type 1 also encodes the Rev protein, which acts through an RNA stem-loop called the Rev-response element to transport unspliced and singly spliced viral RNA species from the nucleus to the cytoplasm. To map the RNA-binding domain of Tat, we performed assays that relied on Rev function using the heterologous RNA-tethering mechanism of Tat and the TAR. By examining the effects of selected targeted mutations of Tat on the abilities of hybrid Tat/Rev proteins to rescue the expression of unspliced mRNA via the TAR, we demonstrated that residues throughout the N-terminal 59 amino acids of Tat are required for binding of Tat and TAR RNA in vivo.

Animals↗

PubMed1998

Plasma membrane-targeted Raf kinase activates NF-kappaB and human immunodeficiency virus type 1 replication in T lymphocytes.

Increasing evidence points to a role of the mitogenic Ras/Raf/MEK/ERK signaling cascade in regulation of human immunodeficiency virus type 1 (HIV-1) gene expression. Stimulation of elements of this pathway leads to transactivation of the HIV-1 promoter. In particular, the NF-kappaB motif in the HIV long terminal repeat (LTR) represents a Raf-responsive element in fibroblasts. Regulation of the Raf kinase in T cells differs from findings with a variety of cell lines that the catalytic domain of Raf (Raf(delta26-303)) shows no activity. In this study, we restored the activity of the kinase in T cells by fusing its catalytic domain to the CAAX motif (-Cx) of Ras, thus targeting the enzyme to the plasma membrane. Constitutive activity of Raf was demonstrated by phosphorylation of mitogen-activated protein kinase kinase (MEK) and endogenous mitogen-activated protein kinase 1/2 (ERK1/2) in A3.01 T cells transfected with Raf(delta26-303)-Cx. Membrane-targeted Raf also stimulates NF-kappaB, as judged by kappaB-dependent reporter assays and enhanced NF-kappaB p65 binding on band shift analysis. Moreover, we found that active Raf transactivates the HIV(NL4-3) LTR in A3.01 T lymphocytes and that dominant negative Raf (C4) blocked 12-O-tetradecanoylphorbol-13-acetate induced transactivation. When cotransfected with infectious HIV(NL4-3) DNA, membrane-targeted Raf induces viral replication up to 10-fold over basal levels, as determined by the release of newly synthesized p24gag protein. Our study clearly demonstrates that the activity of the catalytic domain of Raf in A3.01 T cells is dependent on its cellular localization. The functional consequences of active Raf in T lymphocytes include not only NF-kappaB activation and transactivation of the HIV(NL4-3) LTR but also synthesis and release of HIV particles.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1995

Potent inhibition of human immunodeficiency virus type 1 (HIV-1) replication by inducible expression of HIV-1 PR multimers.

Constructs were generated in which expression of human immunodeficiency virus type 1 (HIV-1) protease (PR) was placed under control of the HIV-1 long terminal repeat, thus requiring the HIV-1 Tat protein for expression of PR. The activity of PR was assessed by cotransfection with a construct producing a Gag substrate. Expression of PR as an intramolecular multimer resulted in a large increase in PR activity in comparison with the level obtained with the expression of PR as a monomer. A cytotoxic effect of PR expression was also exhibited by the constructs expressing PR multimers. CD4+ T-cell lines were generated with a construct producing PR as a linked tetramer and screened for PR activity and inducibility. The replication of HIV-1 in these cell lines was several orders of magnitude reduced in comparison with that in cell lines not expressing PR. Infection in these cell lines could be detected early after infection but disappeared over time. Infection of the PR-expressing cell lines could be increased several orders of magnitude by the addition of a specific inhibitor of PR, U75875 (Upjohn), after infection of the cells, demonstrating that the potent inhibition of HIV-1 replication in these cells was directly due to the expression of PR.

Amino Acid Sequence↗

PubMed1989

Emergence of large plaque-producing clones of human immunodeficiency virus (HIV) in vitro.

Large plaque-inducing clones were obtained from small plaque-inducing parental clones of human immunodeficiency virus (HIV) by the plaque-cloning method. The cloned HIVs that formed large and small plaques were studied as follows: 1) infectivity was determined by the ratio of plaque-forming units (PFU) to reverse transcriptase (RT) activity; 2) viral growth was assessed by the amount (RT activity) of virus after infection; and 3) HIV long terminal repeat (LTR)-linked gene expression of the viruses was measured by chloramphenicol acetyltransferase (CAT) assay using persistently infected MOLT-4 cells. Results showed that clones producing large plaques showed similar or slightly lower infectivity but higher virus production, faster viral growth, and higher gene expression activity than clones producing small plaques. These analyses revealed that clones producing large plaques could replicate more rapidly than those producing small plaques. Restriction enzyme map analysis of these cloned viruses showed that they were also genetically different. These results suggest that the changes in the biological features observed here might be due to mutation during the cloning procedure.

DNA, Viral↗

PubMed1989

Activity of synthetic peptides from the Tat protein of human immunodeficiency virus type 1.

To determine which of the 86 amino acids in the Tat protein of human immunodeficiency virus type 1 (HIV-1) are important for transactivation, peptides from Tat were synthesized and their activity was measured in cells containing a chloramphenicol acetyltransferase reporter gene under control of the HIV long terminal repeat promoter. Although the Tat sequence contains arginine- and cysteine-rich stretches that are difficult to synthesize, it was possible to prepare pure peptides in good yield by using fluoren-9-ylmethoxycarbonyl (Fmoc) chemistry. A peptide containing residues 1-58 had 5-10% the activity of full-length Tat. Deleting 4 amino acids from the N terminus of this peptide further reduced activity, while peptides with more extensive N-terminal deletions and peptides missing the basic region at the C terminus had no detectable activity. A peptide previously reported to transactivate, Tat-(37-62), was completely inactive in our assays. Inactive peptides were also tested as possible inhibitors of transactivation. Tat-(21-38), which contains the cysteine-rich region and can form heterodimers with intact Tat in vitro, showed inhibition at high peptide concentrations. However, this effect was not specific for Tat or for the HIV promoter, since the peptide also inhibited expression from the simian virus 40 early promoter.

Amino Acid Sequence↗

PubMed1998

Optimization of polymerase chain reaction for detection of HIV type 2 DNA.

The aim of this study was to increase the sensitivity of an earlier version of an HIV-2 nested PCR assay based on primers in the gag, pol, LTR, and env regions. The assay was first optimized with regard to concentrations of dNTP, MgCl2, and primers, using a method that allowed optimization of all three parameters in only two test runs. We then designed and optimized new primer sets in the LTR, gag, and gag/pol regions that were based on more isolates than were the former (old) primer sets. Samples from 57 HIV-2 antibody-positive individuals were tested with the four old primer sets as well as with the three new primer sets. Five primer sets from this run (new gag, new gag/pol, old LTR, old env, and new LTR) were then tested with 35 more samples, giving a total number of 92 tested samples from HIV-2-infected individuals. At initial testing of the 92 samples a combination of primer sets from two different regions yielded a sensitivity ranging from 93.5 to 98.9%. After repeated testing the sensitivity ranged from 96.7 to 100% for the different primer combinations. The specificity was 100% for all primer sets except old LTR, which had a specificity of 97%. In conclusion, it is possible to create a more sensitive PCR assay by optimizing the different PCR parameters as well as by including primer sets based on more isolates.

DNA Primers↗

PubMed2006

A high-throughput drug screening system for HIV-1 transcription inhibitors.

Identification of HIV-1 transcription inhibitors was previously performed using infectivity assays. As de novo HIV-1 infection is highly sensitive to even minor compound toxicities, these assays are plagued by extremely high levels of false-positive hits. Hit identification is further complicated because infectivity assays lack target specificity. The authors demonstrate that it is possible to overcome these limitations by establishing a stable, chronically actively HIV-1-infected reporter cell line that exclusively identifies HIV-1 transcription inhibitors. In the reporter cell line, 2 spectrally separated fluorescence proteins serve as simultaneously accessible quantitative markers of HIV-1 expression and drug toxicity. The combined analysis of these markers drastically reduces the level of false-positive hits. As determination of fluorescence intensity in a plate-based format can be performed in a noninvasive manner, repeated measurements of fluorescence levels over several days after compound addition can be used to define the kinetic and dynamic characteristics of inhibitory drug effects. In addition, because of the stable nature of the reporter cell line, the assay requires no cell manipulation during assay preparation or assay analysis, rendering the system extremely cost-effective and reliable.

Animals↗

PubMed1997

Human immunodeficiency virus type 1 nucleocapsid protein promotes efficient strand transfer and specific viral DNA synthesis by inhibiting TAR-dependent self-priming from minus-strand strong-stop DNA.

During the first strand transfer in reverse transcription, minus-strand strong-stop DNA [(-) SSDNA] is annealed to the 3' end of the acceptor RNA in a reaction mediated by base-pairing between terminal repeat sequences in the RNA and their complement in the DNA. The large stem-loop structure in the repeat region known as TAR could interfere with this annealing reaction. We have developed an in vitro human immunodeficiency virus type 1 (HIV-1) system to investigate the effect of TAR on strand transfer. Mutational analysis demonstrates that the presence of TAR in the donor and acceptor templates inhibits strand transfer and is correlated with extensive synthesis of heterogeneous DNAs formed by self-priming from (-) SSDNA. These DNAs are not precursors to the transfer product. Interestingly, products of self-priming are not detected in HIV-1 endogenous reactions; this suggests that virions contain a component which prevents self-priming. Our results show that the viral nucleocapsid protein (NC), which can destabilize secondary structures, drastically reduces self-priming and dramatically increases the efficiency of strand transfer. In addition, the data suggest that the ability to eliminate self-priming is a general property of NC which is manifested during reverse transcriptase pausing at sites of secondary structure in the template. We conclude that this activity of NC is critical for achieving highly efficient and specific viral DNA synthesis. Our findings raise the possibility that inactivation of NC could provide a new approach for targeting reverse transcription in anti-HIV therapy.

DNA, Single-Stranded↗

PubMed1994

The influence of DNA and nucleosome structure on integration events directed by HIV integrase.

DNA copies of the human immunodeficiency virus (HIV) genome integrate nonrandomly into the chromosomal DNA of the host cell. In this report, we investigate the molecular basis of this selectivity using the virus-encoded HIV integrase to direct integration of a synthetic HIV long terminal repeat substrate into either DNA molecules of known structure or previously defined nucleosomal complexes. We find that the structure of the target greatly influences the site of integration, and, moreover, DNA curvature, flexibility, and rigidity in solution all influence the frequency of integration. Importantly, for DNA with all of these properties, the distortion of the double helix directed by association with the histone proteins promotes the integration reaction and alters the distribution of sites that are selected for integration. We suggest that both intrinsic DNA structure and the folding of DNA into chromosomal structures will exert a major influence on target site selection for integration of the viral genome.

Base Sequence↗

PubMed1993

Double-stranded strong-stop DNA and the second template switch in human immunodeficiency virus (HIV) DNA synthesis.

Synthesis of unintegrated retroviral DNA via reverse transcription is thought to involve two separate template switches. Minus-strand strong-stop DNA, a major product of in vitro reactions using detergent-treated virions, is synthesized prior to the first template switch. Plus-strand strong-stop DNA, which is found base-paired to near full-length minus-strand DNA in infected cells, is believed to be synthesized before the second template switch. Using a synchronized, one-step HIV infection model, we report here the detection in acutely infected cells of a novel double-stranded strong-stop HIV DNA with a discrete length of approximately 650 base pairs, commencing at or near the left hand end of the right-hand U3 region of the HIV long terminal repeat. The plus-strand of this double-stranded strong-stop DNA possesses the primer binding site sequence and appears to be synthesized prior to the completion of the synthesis of its complementary minus-strand. In contrast, the minus-strand of the double-stranded strong-stop DNA lacks the primer binding site sequence after RNaseH digestion. We propose that a transient free plus-strand strong-stop DNA is released from its template by displacement synthesis and subsequently used as template for the synthesis of its complementary minus-strand. The proposed transient free plus-strand strong-stop DNA may also mediate the second template switch.

Base Sequence↗