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At least 505 records · Page 28Linked to original sources
PubMed1994

Identification of a human immunodeficiency virus type 1 TAR binding protein in human hepatoblastoma HepG2 cells that trans-activates HIV-1 LTR-directed gene expression.

Recently, we have shown that the human immunodeficiency virus (HIV-1) long terminal repeat (LTR) directed chloramphenicol acetyltransferase (CAT) gene is efficiently expressed in human hepatoblastoma HepG2 cells and these cells can support productive HIV-1 replication. In this study we show that HepG2 cells contain a nuclear factor that binds to the HIV-1 trans-activating region (TAR), which we named HepG2-derived TAR binding protein (HTBP). Gel retardation assays using synthetic oligonucleotide probes carrying different mutations in the TAR region and competition DNA mobility-shift experiments using these oligonucleotides revealed the binding site encompassing between +7 and +13 nucleotides (5'-TCTGGTT-3') in the HIV-1 LTR. An in vivo CAT competition assay using -65HIV-1 LTR CAT as a reporter plasmid and various competitor plasmids containing these mutated oligonucleotides also demonstrated that HTBP can influence the HIV-1 LTR-directed CAT gene expression in HepG2 cells by interaction with a specific sequence in the TAR region.

Base Sequence↗
PubMedDate not supplied

The contribution of low tar cigarettes to environmental tobacco smoke.

A series of low tar cigarettes (LTC) were smoked and the quantities of condensable mainstream (inhaled) and sidestream (between puffs) smoke compounds were determined and compared to those produced by a high tar, nonfilter cigarette. It was found that the LTC produced large quantities of sidestream smoke condensates, about equal to the high tar cigarette, and contained very high levels of toxic or cocarcinogenic phenols. On an equal weight basis, the LTC emitted more of these hazardous compounds into sidestream and environmental tobacco smoke. Higher smoke yields of a flavor additive and a sugar degradation product indicated addition of such compounds during the manufacture of LTC. It was concluded that, compared to a high tar cigarette, smoking LTC may be better for the smoker, but not for the nearby nonsmoker. Information should be developed to allow smokers to choose LTC that produce lower levels of hazardous compounds in their environmentally emitted sidestream smoke.

Chromatography, Gas↗
PubMed1998

Modified (PNA, 2'-O-methyl and phosphoramidate) anti-TAR antisense oligonucleotides as strong and specific inhibitors of in vitro HIV-1 reverse transcription.

Natural beta-phosphodiester 16mer and 15mer antisense oligonucleotides targeted against the HIV-1 and HIV-2 TAR RNAs respectively were previously described as sequence-specific inhibitors of in vitro retroviral reverse transcription. In this work, we tested chemically modified oligonucleotide analogues: alpha-phosphodiester, phosphorothioate, methylphosphonate, peptide nucleic acid or PNA, 2'- o -methyl and (N3'-P5') phosphoramidate versions of the 16mer anti-TAR oligonucleotide. PNA, 2'- O -methyl and (N3'-P5') phosphoramidate oligomers showed a strong inhibitory effect compared with the unmodified 16mer, with reverse transcription inhibition (IC50) values in the nanomolar range. The inhibition was sequence-specific, as scrambled and mismatched control oligonucleotides were not able to inhibit cDNA synthesis. No direct binding of the 2'- O -methyl, PNA or (N3'-P5') phosphoramidate anti-TAR oligonucleotides to the HIV-1 reverse transcriptase was observed. The higher T m obtained with 2'- O -methyl, (N3'-P5') phosphoramidate and PNA molecules concerning the annealing with the stem-loop structure of the TAR RNA, in comparison with the beta-phosphodiester oligonucleotides, is correlated with their high inhibitory effect on reverse transcription.

Anti-HIV Agents↗
PubMed2001

Mutations in the TAR hairpin affect the equilibrium between alternative conformations of the HIV-1 leader RNA.

The HIV-1 untranslated leader RNA can adopt two mutually exclusive conformations that represent alternative secondary structures. This leader RNA can fold either an extended duplex through long-distance base pairing or a branched conformation in which the RNA locally folds into hairpin structures. Both leader RNA conformations have the TAR hairpin in common, which forms the extreme 5' end of all HIV-1 transcripts. We report that truncation of the TAR hairpin shifts the equilibrium between the two RNA conformations away from the thermodynamically favored long-distance interaction. However, the equilibrium is partially restored in response to the cations Na(+) and Mg(2+). The transcripts with mutant TAR structures allowed us to investigate conditions affecting the competition between the alternative conformations of the HIV-1 leader RNA. We also demonstrate that the change in conformation of the leader RNA due to TAR truncations severely affects formation of the HIV-1 RNA dimer.

Base Pairing↗
PubMed2003

Zinc finger-dependent HIV-1 nucleocapsid protein-TAR RNA interactions.

In the minus-strand transfer step of HIV-1 reverse transcription, the nucleocapsid protein (NC) promotes annealing of the 3' 'R' (repeat) region of the RNA genome to its complementary sequence located in the newly synthesized minus-strand strong-stop DNA. The R region contains the highly stable transactivation response (TAR) RNA hairpin. To gain insights into the molecular details of TAR RNA-NC interactions, we carried out hydroxyl radical footprinting, as well as gel-shift and fluorescence anisotropy binding assays using wild-type and mutant forms of NC. Our results support the conclusion that NC variants with mutations in their zinc finger domains have dramatically altered TAR RNA binding interactions relative to wild-type NC. These data demonstrate that a specific zinc finger architecture is required for optimal TAR RNA binding, and help to explain the requirement for the zinc finger motifs of NC in its role as a nucleic acid chaperone in minus-strand transfer.

Base Sequence↗
PubMed2005

Monitoring tat peptide binding to TAR RNA by solid-state 31P-19F REDOR NMR.

Complexes of the HIV transactivation response element (TAR) RNA with the viral regulatory protein tat are of special interest due in particular to the plasticity of the RNA at this binding site and to the potential for therapeutic targeting of the interaction. We performed REDOR solid-state NMR experiments on lyophilized samples of a 29 nt HIV-1 TAR construct to measure conformational changes in the tat-binding site concomitant with binding of a short peptide comprising the residues of the tat basic binding domain. Peptide binding was observed to produce a nearly 4 A decrease in the separation between phosphorothioate and 2'F labels incorporated at A27 in the upper helix and U23 in the bulge, respectively, consistent with distance changes observed in previous solution NMR studies, and with models showing significant rearrangement in position of bulge residue U23 in the bound-form RNA. In addition to providing long-range constraints on free TAR and the TAR-tat complex, these results suggest that in RNAs known to undergo large deformations upon ligand binding, 31P-19F REDOR measurements can also serve as an assay for complex formation in solid-state samples. To our knowledge, these experiments provide the first example of a solid-state NMR distance measurement in an RNA-peptide complex.

Binding Sites↗
PubMed2006

Dynamics of nascent mRNA folding and RNA-protein interactions: an alternative TAR RNA structure is involved in the control of HIV-1 mRNA transcription.

HIV-1 Tat protein regulates transcription elongation by binding to the 59 nt TAR RNA stem-loop structure transcribed from the HIV-1 5' long terminal repeat (5'-LTR). This established Tat-TAR interaction was used to investigate mRNA folding and RNA-protein interactions during early transcription elongation from the HIV-1 5'-LTR. Employing a new site-specific photo-cross-linking strategy to isolate transcription elongation complexes at early steps of elongation, we found that Tat interacts with HIV-1 transcripts before the formation of full-length TAR (TAR59). Analysis of RNA secondary structure by free energy profiling and ribonuclease digestion indicated that nascent transcripts folded into an alternative TAR RNA structure (TAR31), which requires only 31 nt to form and includes an analogous Tat-binding bulge structure. Functionally, TAR31, similar to TAR59, acts as a transcriptional terminator in vitro, and mRNA expression from TAR31-deficient HIV-1 5'-LTR mutant promoters is significantly decreased. Our results support a role for TAR31 in the control of HIV-1 mRNA transcription and we propose that this structure is important to stabilize the short early transcripts before the transcription complex commits for processive elongation. Overall, this study demonstrates that RNA folding during HIV-1 transcription is dynamic and that as the nascent RNA chain grows during transcription, it folds into a number of conformations that function to regulate gene expression. Finally, our results provide a new experimental strategy for studying mRNA conformation changes during transcription that can be applied to investigate the folding and function of nascent RNA structures transcribed from other promoters.

Gene Products, tat↗
PubMed1999

Preparation of HIV TAR RNA with RNA scissors.

Two hammerhead ribozymes derived from plant pathogenic RNAs were used to cut off the HIV TAR RNA from the T7 RNA transcript through a cis cleavage reaction. Stem I of the (+)vLTSV ribozyme comprises 8 nucleotides of the 5' terminus of TAR RNA, but stem III of the (+)sTRSV ribozyme consists of 8 nucleotides of the 3' end of TAR RNA. The construct containing two GUC hammerhead ribozyme target sequences identified the cleavage sites to cut off a required RNA molecule. This method was applied for preparation of 35 nt long TAR RNA. Its activity was proved by the complex formation with the Tat protein. It seems that this approach based on RNA scissors can also be used for the generation of required RNA molecules, RNA decoys or RNA aptamers in vivo.

Bacteriophage T7↗
PubMedDate not supplied

Smoke yields of tobacco-specific nitrosamines in relation to FTC tar level and cigarette manufacturer: analysis of the Massachusetts Benchmark Study.

OBJECTIVES: This research assessed the relationship between the deliveries of carcinogenic tobacco-specific nitrosamines (TSNAs) and the Federal Trade Commission (FTC) "tar" ratings of US commercial cigarettes. METHODS: Analysis of covariance (ANCOVA) was used to assess the explanatory power of FTC tar, the particular manufacturer, and other cigarette characteristics to predict the yields of four TSNAs (N'-nitrosonornicotine [NNN], 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone [NNK], N'-nitrosoanatabine [NAT], and N'-nitrosoanabasine [NAB]) in 26 US commercial brands tested in the 1999 Massachusetts Benchmark Study. RESULTS: When FTC tar alone was used to predict TSNA yield, the squared correlation coefficient (R(2)) was only 38% for NNN, 76% for NNK, 46% for NAT, and 49% for NAB. Inclusion of manufacturer-specific variables significantly (p < 0.001) increased the estimated R(2) for three of the four species of nitrosamine to: 78% for NNN, 88% for NNK, and 81% for NAT. Inclusion of other cigarette characteristics (filter type, paper permeability, tobacco weight, tip dilution) did not reduce the significance of the manufacturer-specific effects. Federal Trade Commission nicotine and carbon monoxide (CO) yields were no better at predicting TSNA levels. CONCLUSIONS: FTC ratings for tar, nicotine, and carbon monoxide do not tell the entire story about the comparative yields of toxic agents in marketed cigarette brands. The significant manufacturer-specific effects suggest that proprietary blending and processing of tobacco matter as well. Public, brand-by-brand disclosure of the yields of TSNA and possibly other smoke constituents appears to be warranted.

Analysis of Variance↗
PubMed1997

TAR and Sp1-independent transactivation of HIV long terminal repeat by the Tat protein in the presence of human cytomegalovirus IE1/IE2.

OBJECTIVE: The HIV Tat protein is a transcriptional transactivator of the HIV-1 long terminal repeat (LTR) promoter element. Its activity depends on its direct interaction with the trans-activation response (TAR) element, although TAR-independent activation by Tat has been demonstrated in different cells. Herpesviruses in general and human cytomegalovirus (HCMV) in particular are often isolated from HIV-1-infected patients and could play a role in the activation of latent HIV and in a subsequent increase in HIV replication. HCMV immediate early gene products (IE1 and IE2) are nuclear phosphoproteins that play a pivotal role in HCMV replication and have been shown to transregulate both viral and cellular gene expression. It has repeatedly been shown that HCMV IE1/IE2 can independently transactivate HIV-1 LTR. The aim of this study was to investigate IE1/IE2 transactivation of HIV-1 LTR in a CD4+ T-cell line in the absence and presence of HIV-1 Tat to establish whether IE1/IE2 can synergize with Tat. METHODS: HIV-1 LTR transactivation by HCMV IE1/IE2 in the presence and absence of HIV-1 Tat was determined by transient transfection experiments of J-Jhan lymphoblastoid cells with a series of different expression vectors. RESULTS: We found a strong synergistic transactivation between HIV Tat and the IE1-IE2 complex on HIV LTR activity using vectors driven either by wild-type LTR or by the nuclear factor NF-kappa(B) response element-mutated HIV LTR. IE1/IE2 synergism with HIV Tat was also observed in Sp1 binding site-mutated for TAR-deleted LTR, which cannot be activated by Tat alone. This cooperation is abolished when the region in IE2 that binds the TATA box binding protein is deleted. CONCLUSIONS: The results obtained indicate that Sp1-binding and TAR sequences are not strictly required for Tat responsiveness when Tat is directed to the HIV promoter by HCMV IE1-IE2. This synergistic effect is mediated by the IE2 and TATA-binding region, and could play a major role in HIV activation when cells are infected by both viruses, a feature often observed in AIDS patients.

Cytomegalovirus↗
PubMed1983

The Rh antigen Tar (Rh40) causing haemolytic disease of the newborn.

Haemolytic disease of the newborn (HDN), in the fifth child of an Israeli of Moroccan origin, disclosed anti-Tar in the serum of the mother. This anti-Tar, the second reported example, is the first to cause HDN and was stimulated by previous pregnancies. Tests on blood from the family show that the Tar antigen in this family is associated with a CDe complex which gives rise to a weak expression of the D antigen. A study of 1327 donors in Jerusalem revealed one Tar + propositus.

Coombs Test↗
PubMed1994

Transmembrane signalling by the chimeric chemosensory receptors of Escherichia coli Tsr and Tar with heterologous membrane-spanning regions.

The serine and aspartate chemosensory receptors (Tsr and Tar) of Escherichia coli have two membrane-spanning regions TM1 and TM2. To investigate their roles in transmembrane signalling, we constructed two chimeric receptors from Tsr and Tar with heterologous combinations of TM1 and TM2: the N-terminus of one receptor, including TM1 and the periplasmic domain, was fused to the C-terminus of the other, beginning with TM2. Both of the chimeric receptor genes rescued the chemotactic defect of a receptorless E. coli strain, indicating that the chimeric receptors are functional. Their apparent affinities for the specific ligands were the same as those of Tsr or Tar. Therefore, as far as transmembrane signalling abilities are concerned, the TM2 regions of Tsr and Tar are interchangeable, suggesting that sequence-specific interaction between TM1 and TM2 may not be required for the signal transmission across the membrane. The cells expressing either of the chimeric receptors, however, showed 'smooth', biased, basal swimming patterns. Moreover, they adapted quickly after stimulation with the repellent glycerol. This rapid adaptation was observed even in the methyltransferase-defective strain. Therefore, exchange of TM2 might impose structural constraints on the chimeric receptors that stabilize conformations which elicit smooth swimming.

Adaptation, Physiological↗
PubMed1992

Conformation of the TAR RNA-arginine complex by NMR spectroscopy.

The messenger RNAs of human immunodeficiency virus-1 (HIV-1) have an RNA hairpin structure, TAR, at their 5' ends that contains a six-nucleotide loop and a three-nucleotide bulge. The conformations of TAR RNA and of TAR with an arginine analog specifically bound at the binding site for the viral protein, Tat, were characterized by nuclear magnetic resonance (NMR) spectroscopy. Upon arginine binding, the bulge changes conformation, and essential nucleotides for binding, U23 and A27.U38, form a base-triple interaction that stabilizes arginine hydrogen bonding to G26 and phosphates. Specificity in the arginine-TAR interaction appears to be derived largely from the structure of the RNA.

Arginine↗
PubMed1990

Fragments of the HIV-1 Tat protein specifically bind TAR RNA.

Proteolytically produced carboxyl-terminal fragments of the human immunodeficiency virus type-1 (HIV-1) Tat protein that include a conserved region rich in arginine and lysine bind specifically to transactivation response RNA sequences (TAR). A chemically synthesized 14-residue peptide spanning the basic subdomain also recognizes TAR, identifying this subdomain as central for RNA interaction. TAR RNA forms a stable hairpin that includes a six-residue loop, a trinucleotide pyrimidine bulge, and extensive duplex structure. Competition and interference experiments show that the Tat-derived fragments bind to double-stranded RNA and interact specifically at the pyrimidine bulge and adjacent duplex of TAR.

Amino Acid Sequence↗
PubMed1985

Genetics of methyl-accepting chemotaxis proteins in Escherichia coli: null phenotypes of the tar and tap genes.

The tar and tap genes are located adjacent to one another in an operon of chemotaxis-related functions. They encode methyl-accepting chemotaxis proteins implicated in tactic responses to aspartate and maltose stimuli. The functional roles of these two gene products were investigated by isolating and characterizing nonpolar, single-gene deletion mutants at each locus. Deletions were obtained by selecting for loss or a defective Mu d1 prophage inserted in either the tar or tap gene. The extent of the tar deletions was determined by genetic mapping with Southern hybridization. Representative deletion mutants were surveyed for chemotactic responses on semisolid agar and by temporal stimulation in a tethered cell assay to assess flagellar rotational responses to chemoeffector compounds. The tar deletion strains exhibited complete loss of aspartate and maltose responses, whereas the tap deletion strains displayed a wild-type phenotype under all conditions tested. These findings indicate that the tap function is unable to promote chemotactic responses to aspartate and maltose, and its role in chemotaxis remains unclear.

Bacterial Proteins↗
PubMed1994

Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent activator of long terminal repeat-directed transcription. While in most cell types, activation requires interaction of Tat with the unusual transcription element TAR, astrocytic glial cells support TAR-independent transactivation of HIV-1 transcription by Tat. This alternative pathway of Tat activation is mediated by the viral enhancer, a kappa B domain capable of binding the prototypical form of the transcription factor nuclear factor kappa B (NF-kappa B) present in many cell types, including T lymphocytes. Tat transactivation mediated by the kappa B domain is sufficient to allow replication of TAR-deleted mutant HIV-1 in astrocytes. The present study demonstrates the existence of kappa B-specific binding factors present in human glial astrocytes that differ from prototypical NF-kappa B. The novel astrocyte-derived kappa B-binding activity is retained on an HIV-1 Tat affinity column, while prototypical NF-kappa B from Jurkat T cells is not. In vitro transcription studies demonstrate that astrocyte-derived kappa B-binding factors activate transcription of the HIV-1 long terminal repeat and that this activation is dependent on the kappa B domain. Moreover, TAR-independent transactivation of HIV-1 transcription is reproduced in vitro in an astrocyte factor-dependent manner which correlates with kappa B-binding activity. The importance of the central nervous system-enriched kappa B transcription factor in the regulation of HIV-1 expression is discussed.

Astrocytes↗
PubMed1995

Interaction of nuclear protein p140 with human immunodeficiency virus type 1 TAR RNA in mitogen-activated primary human T lymphocytes.

Several lines of evidence suggest that cellular proteins play a role during human immunodeficiency virus type 1 (HIV-1) Tat-mediated trans activation. A recent report from this laboratory has shown that a 140-kDa HeLa nuclear protein (p140) binds specifically to the lower stem region of the Tat response element, TAR RNA. Since HIV-1 trans activation is most efficient in proliferating T cells, we investigated the binding of p140 to TAR RNA in unstimulated and mitogen-activated, G1-phase primary T lymphocytes. TAR RNA/protein-binding activity was low in resting cells but increased significantly within 2 h of activation and remained elevated for at least 48 h. Corresponding increases in p140 protein levels were observed with most but not all donors, suggesting that an additional nuclear factor(s) may be required for efficient binding of this protein to TAR RNA in activated T cells.

Base Sequence↗
PubMed2002

A minimal chimera of human cyclin T1 and tat binds TAR and activates human immunodeficiency virus transcription in murine cells.

The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5' end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn(2+)-dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn(2+)-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex.

3T3 Cells↗