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PubMed1998

Structural model of the HIV-1 Tat(46-58)-TAR complex.

The trans-activator protein (Tat) of human immunodeficiency virus type 1 (HIV-1) binds to an uridine-rich bulge of an RNA target (TAR; trans-activation responsive element) predominantly via its basic sequence domain. The structure of the Tat(46-58)-TAR complex has been determined by a novel modeling approach relying on structural information about one crucial arginine residue and crosslink data. The strategy described here solely uses this experimental data without additional "modeling" assumptions about the structure of the complex in order to avoid human bias. Model building was performed in a fashion similar to structure calculations from nuclear magnetic resonance (NMR)-spectroscopic data using restrained molecular dynamics. The resulting set of structures of Tat(46-58) in its complex with TAR reveals that all models have converged to a common fold, showing a backbone root mean square deviation (RMSD) of 1.36A. Analysis of the calculated structures suggests that HIV-I Tat forms a hairpin loop in its complex with TAR that shares striking similarity to the hairpin formed by the structure of the bovine immunodeficiency virus Tat protein after TAR binding as determined by NMR studies. The outlined approach is not limited to the Tat-TAR complex modeling, but is also applicable to all molecular complexes with sufficient biochemical and biophysical data available.

Amino Acid Sequence↗
PubMed2004

Incomplete compensation does not imply reduced harm: yields of 40 smoke toxicants per milligram nicotine in regular filter versus low-tar cigarettes in the 1999 Massachusetts Benchmark Study.

The yields of 40 toxic smoke constituents per mg nicotine were computed in three matched pairs of higher- and lower-tar cigarettes tested in the 1999 Massachusetts Benchmark Study. In the three pairs, 19, 20 and 17 compounds, respectively, exhibited significantly greater yields per mg nicotine in the lower-tar than the corresponding higher-tar brand. Based on the assumption that toxicant yields per mg nicotine were independent of smoking intensity, the minimum degree of nicotine compensation necessary to obtain equal or greater dosages of these toxic compounds from the lower-tar cigarette ranged from 0% to 73%. A smoker who compensates only incompletely for nicotine may thus obtain a higher dosage of a specific smoke toxicant from a lower-tar cigarette than from a higher-tar cigarette. From the toxicological standpoint, incomplete compensation for nicotine does not necessarily translate into harm reduction.

Administration, Inhalation↗
PubMed2001

Aromatic polyamidines inhibiting the Tat-induced HIV-1 transcription recognize structured TAR-RNA.

We have investigated the effects of aromatic polyamidines on HIV-1 transcription. We found a block to Tat-induced HIV-1 transcription assessed by inhibition of CAT activity in HL3T1 cells at a concentration lower than the IC50 value, suggesting that molecules with three (TAPB) and four (TAPP) benzamidine rings could be useful against HIV-1. In contrast, aromatic polyamidines with only two benzamidine rings (DAPP) did not block Tat-induced transcription. We reasoned that this effect could be due to binding of TAPB and TAPP to HIV-1 TAR RNA. By EMSA and filter binding assays, we studied possible interactions of aromatic polyamidines with HIV-1 TAR RNA. Wild-type TAR RNA or TAR RNA with mutations in the stem or bulge sequences, but retaining the stem-loop structure, was used to define the RNA-binding activities of these compounds. Our data suggest that aromatic polyamidines with two (DAPP) and four (TAPP) benzamidine rings, respectively, do not bind to TAR RNA or bind without sequence selectivity. Interestingly, an aromatic polyamidine with three benzamidine rings (TAPB) recognizes the wild-type TAR RNA in a specific manner. Furthermore, we found that introduction of one halogen atom into the benzamidine rings strongly increases the RNA-binding activity of these compounds.

Anti-HIV Agents↗
PubMed2003

Interactions of hairpin oligo-2'-O-methylribonucleotides containing methylphosphonate linkages with HIV TAR RNA.

Methylphosphonate-modified oligo-2'-O-methylribonucleotides 15-20 nucleotides (nt) in length were prepared whose sequences are complementary to the 5' and 3' sides of the upper hairpin of HIV trans-acting response element (TAR) RNA. These anti-TAR oligonucleotides (ODNs) form stable hairpins whose melting temperatures (Tm) range from 55 degrees C to 80 degrees C. Despite their rather high thermal stabilities, the hairpin oligo-2'-O-methylribonucleotides formed very stable complexes with TAR RNA, with dissociation constants in the nanomolar concentration range at 37 degrees C. The affinities of the hairpin oligomers for TAR RNA were influenced by the positions of the methylphosphonate linkages. The binding affinity was reduced approximately 17-fold by the presence of two methylphosphonate linkages in the TAR loop complementary region (TLCR) of the oligomer, whereas methylphosphonate linkages outside this region increased binding affinity approximately 3-fold. The configurations of the methylphosphonate linkages in the TLCR also affected binding affinity, with the RpRp isomer showing significantly higher binding than the SpSp isomer. In addition to serving as probes of the interactions between the oligomer and TAR RNA, the presence of the methylphosphonate linkages in combination with the hairpin structure increases the resistance of these oligomers to degradation by exonucleases found in mammalian serum. The combination of high binding affinity and nuclease resistance of the hairpin ODNs containing methylphosphonate linkages suggests their potential utility as antisense compounds.

HIV Long Terminal Repeat↗
PubMed1997

Transfer of Tat and release of TAR RNA during the activation of the human immunodeficiency virus type-1 transcription elongation complex.

The HIV-1 trans-activator protein, Tat, is a potent activator of transcriptional elongation. Tat is recruited to the elongating RNA polymerase during its transit through the trans-activation response region (TAR) because of its ability to bind directly to TAR RNA expressed on the nascent RNA chain. We have shown that transcription complexes that have acquired Tat produce 3-fold more full-length transcripts than complexes not exposed to Tat. Western blotting experiments demonstrated that Tat is tightly associated with the paused polymerases. To determine whether TAR RNA also becomes attached to the transcription complex, DNA oligonucleotides were annealed to the nascent chains on the arrested complexes and the RNA was cleaved by RNase H. After cleavage, the 5' end of the nascent chain, carrying TAR RNA, is quantitatively removed, but the 3' end of the transcript remains associated with the transcription complex. Even after the removal of TAR RNA, transcription complexes that have been activated by Tat show enhanced processivity. We conclude that Tat, together with cellular co-factors, becomes attached to the transcription complex and stimulates processivity, whereas TAR RNA does not play a direct role in the activation of elongation and is used simply to recruit Tat and cellular co-factors.

Bacterial Proteins↗
PubMed1996

The binding mode of drugs to the TAR RNA of HIV-1 studied by electric linear dichroism.

For the first time, the interaction between a series of small molecules and the TAR RNA of HIV-1 has been investigated by electric linear dichroism (ELD). The compounds tested include the DNA intercalating drugs proflavine and ethidium bromide and an amsacrine-4-carboxamide DNA-threading intercalator as well as the AT-specific DNA minor groove binders netropsin, Hoechst 33258, berenil and DAPI. In all cases except for netropsin, negative reduced dichroism signals were measured in the drug absorption band. In agreement with previous studies, the results indicate that both classical and threading intercalation can occur with the TAR RNA. The ELD data show that the mode of binding of the drugs Hoechst 33258, berenil and DAPI to the TAR RNA is similar to their binding mode in GC-rich regions of DNA and likely involves intercalation into the A-form TAR RNA helix. The wide and shallow minor groove of the TAR RNA is apparently not accessible to DNA minor groove binding drugs such as netropsin. The ELD technique appears uniquely valuable as a means of investigating the interaction of drugs with the TAR RNA.

Antiviral Agents↗
PubMed1997

TAR RNA decoys inhibit tat-activated HIV-1 transcription after preinitiation complex formation.

The ability of the HIV-1 Tat protein to trans -activate HIV-1 transcription in vitro is specifically inhibited by a circular TAR RNA decoy. This inhibition is not overcome by adding an excess of Tat to the reaction but is partially overcome by adding Tat in combination with nuclear extract, suggesting that TAR RNA might function by interacting with a complex containing Tat and cellular factor(s). A cell-free transcription system involving immobilized DNA templates was used to further define the factor(s) that interact with TAR RNA. Preinitiation complexes formed in the presence or absence of Tat were purified on immobilized templates containing the HIV-1 promoter. After washing, nucleotides and radiolabelled UTP were added and transcription was measured. The presence of Tat during preinitiation complex formation resulted in an increase in the level of full-length HIV-1 transcripts. This Tat-activated increase in HIV-1 transcription was not inhibited by circular TAR decoys added during preinitiation complex formation but was inhibited by circular TAR decoys subsequently added during the transcription reaction. These results suggest that TAR decoys inhibit Tat-activated HIV-1 transcription after preinitiation complex formation, perhaps by interacting with components of transcription complexes.

Gene Expression Regulation, Viral↗
PubMed1997

Binding of Hoechst 33258 to the TAR RNA of HIV-1. Recognition of a pyrimidine bulge-dependent structure.

The transactivation response region (TAR) RNA is an essential component in transcriptional regulation of the human immunodeficiency virus type-1 (HIV-1) genome. We have examined the interaction between TAR RNA and the bisbenzimidazole derivative Hoechst 33258. Previous studies have shown that this drug, which is well known as an AT-selective DNA minor groove binder, can also interact with GC-rich sequences in DNA as well as with RNA, possibly by intercalation. Absorption spectroscopy, circular dichroism and electric linear dichroism, as well as RNase A footprinting, were employed to compare binding of Hoechst 33258 to wild-type RNA and its analogue lacking the pyrimidine bulge. The uridine bulge, which is an important contributor to the structural stability of TAR, plays an essential role in drug binding. Deletion of the bulge destabilizes both free and drug-bound forms of TAR and markedly affects the orientation of the drug chromophore complexed with the RNA. According to the linear dichroism data, the bisbenzimidazole is oriented more or less perpendicular to the RNA helix axis. The data are compatible with a model in which the bisbenzimidazole chromophore is inserted into the existing cavity created by the pyrimidine bulge. The footprinting experiments, showing that the drug binds to a unique site opposite the unpaired uridine residues, also support this model. The binding of Hoechst 33258 to the sequence 5'-GCUCU, which delimits the cavity, reflects the greater accessibility of that region compared with other sites in the RNA molecule. The identification of a binding site for small molecules in TAR offers promising perspectives for developing drugs that would block the access of TAR RNA to proteins and therefore for the design of anti-HIV agents.

Absorption↗
PubMed1997

Optimal Tat-mediated activation of the HIV-1 LTR promoter requires a full-length TAR RNA hairpin.

HIV-1 transcription from the LTR promoter is activated by the viral Tat protein through interaction with the nascent TAR RNA hairpin structure. The mechanism of Tat-mediated transcriptional activation has been extensively investigated with LTR-CAT reporter genes in transient transfections and, more recently, in infection experiments with mutant HIV-1 variants. Several discrepancies between these two assay systems have been reported. For instance, whereas opening of the lower part of the TAR RNA stem does not affect the promoter activity of an LTR-CAT plasmid in transient assays, the corresponding virus mutant is fully replication-impaired. With the aim to resolve this controversy, we have examined the activity of a set of TAR RNA mutants in transient transfection experiments with a variety of cell types. We now demonstrate that truncated TAR motifs exhibit a severe, but cell-type dependent transcription defect. Whereas full LTR activity is measured in COS cells that have been used regularly in previous transfection assays, a severe defect is apparent in a variety of human cell lines, including T cell lines that are typically used in HIV-1 replication studies. These results suggest the presence of a human protein that participates in Tat-mediated transcriptional activation through binding to the lower part of the TAR stem. Several candidate co-factors have been reported in literature. This study resolves the discrepancy between transfection and infection studies on the requirements of the lower TAR stem structure. The evidence also implies that LTR transcription studies should be performed preferentially in human cell types.

Animals↗
PubMed2001

Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning.

The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest. To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region. For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning. When vectors with a specific hook that was >/=60 bp were utilized, approximately 2% of transformants contained circular YACs with the Tg.AC transgene sequences. Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less. Thus, the minimal length of a unique sequence required for gene isolation by TAR is approximately 60 bp. No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.

Animals↗
PubMed2006

TAR-RNA recognition by a novel cyclic aminoglycoside analogue.

The formation of the Tat-protein/TAR-RNA complex is a crucial step in the regulation of human immunodeficiency virus (HIV)-gene expression. To obtain full-length viral transcripts the Tat/TAR complex has to recruit the positive transcription elongation factor complex (P-EFTb), which interacts with TAR through its cyclin T1 (CycT1) component. Mutational studies identified the TAR hexanucleotide loop as a crucial region for contacting CycT1. Interfering with the interaction between the Tat/CycT1 complex and the TAR-RNA is an attractive strategy for the design of anti-HIV drugs. Positively charged molecules, like aminoglycosides or peptidomimetics, bind the TAR-RNA, disrupting the Tat/TAR complex. Here, we investigate the complex between the HIV-2 TAR-RNA and a neooligoaminodeoxysaccharide by NMR spectroscopy. In contrast to other aminoglycosides, this novel aminoglycoside analogue contacts simultaneously the bulge residues required for Tat binding and the A35 residue of the hexanucleotide loop. Upon complex formation, the loop region undergoes profound conformational changes. The novel binding mode, together with the easy accessibility of derivatives for the neooligoaminodeoxysaccharide, could open the way to the design of a new class of TAR-RNA binders, which simultaneously inhibit the formation of both the Tat/TAR binary complex and the Tat/TAR/CycT1 ternary complex by obstructing both the bulge and loop regions of the RNA.

Aminoglycosides↗
PubMed1989

Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.

The human immunodeficiency virus (HIV-1)-encoded trans-activator (tat) increases HIV gene expression and replication. Previously, we demonstrated that tat facilitates elongation of transcription through the HIV-1 long terminal repeat (LTR) and that short transcripts corresponding to prematurely terminated RNA are released and accumulate in the absence of tat. Here, using a transient expression assay, we tested clustered and compensatory mutations, as well as 3' deletions, in the trans-acting responsive region (tar) and observed that the primary sequence in the loop and secondary structure in the stem of the stem-loop in tar are required for trans-activation by tat. Insertions in the 5' region of tar revealed that tar must be near the site of HIV-1 initiation of transcription for trans-activation by tat. Deletions (3') and an insertion in tar demonstrated that an intact stem-loop is required for the recovery of prematurely terminated transcripts. Short and full-length transcripts were observed also with HIV type 2 (HIV-2) in the absence and presence of tat, respectively. We conclude that an intact stem-loop in tar is essential for trans-activation by tat and that initiation of transcription by HIV-1 promoter factors and elongation of transcription by tat are coupled.

Chloramphenicol O-Acetyltransferase↗
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↗
PubMed1993

The full-length Tat protein is required for TAR-independent, posttranscriptional trans activation of human immunodeficiency virus type 1 env gene expression.

Tat is a protein that dramatically increases the expression of all genes expressed from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat through interaction with a cis-acting target sequence referred to as TAR (for trans-acting responsive region). The tat gene is divided into two coding exons which, when translated, result in the synthesis of an 86-amino-acid protein. However, the 72-amino-acid segment encoded by the first coding exon of tat is sufficient to encode a fully active Tat protein in known assays. We examined expression of the env gene from an LTR that lacks TAR (designated dTAR-env). Surprisingly, only the full-length Tat peptide trans activated expression of the env gene from dTAR-env. Comparison of RNA and protein expression of the env gene in the presence of Tat indicated that the mechanism of trans activation is posttranscriptional rather than transcriptional. To test whether the TAR-independent Tat function is specific to the HIV-1 env gene, we analyzed expression of heterologous genes from the long terminal repeat lacking TAR. These heterologous genes were not trans activated by Tat in the absence of a TAR element, which suggests that the second-exon peptide of Tat has a sequence-specific role in TAR-independent trans activation of the HIV-1 env gene. Analysis of a mutant in the 5' end of the env gene was used to identify a cis-acting sequence required for Tat responsiveness.

Base Sequence↗
PubMed2000

The human immunodeficiency virus type 1 TAR RNA upper stem-loop plays distinct roles in reverse transcription and RNA packaging.

The human immunodeficiency virus type 1 (HIV-1) RNA genome is flanked by a repeated sequence (R) that is required for HIV-1 replication. The first 57 nucleotides of R form a stable stem-loop structure called the transactivation response element (TAR) that can interact with the virally encoded transcription activator protein, Tat, to promote high levels of gene expression. Recently, we demonstrated that TAR is also important for efficient HIV-1 reverse transcription, since HIV-1 mutated in the upper stem-loop of TAR showed a reduced ability both to initiate and to complete reverse transcription. We have analyzed a series of HIV-1 mutant viruses to better defined the structural or sequence elements required for natural endogenous reverse transcription and packaging of virion RNA. Our results indicate that the requirement for TAR in reverse transcription is conformation dependent, since mutants with mutations that alter the upper stem-loop orientation are defective for reverse transcription initiation and have minor defects in RNA packaging. In contrast, TAR mutations that allowed the formation of alternative upper stem-loop structure greatly reduced RNA packaging but did not affect reverse transcription efficiency. These results are consistent with direct involvement of the upper stem-loop structure in packaging of genomic RNA and suggest that the TAR RNA stem-loop from nucleotide +18 to +42 interacts with other components of the reverse transcription initiation complex to promote efficient reverse transcription.

Base Pairing↗
PubMed2000

Binding of Tat to TAR and recruitment of positive transcription elongation factor b occur independently in bovine immunodeficiency virus.

Transcriptional transactivators (Tat) from many lentiviruses interact with their cognate transactivation response RNA structures (TAR) to increase rates of elongation rather than initiation of transcription. For several of them, the complex of Tat and a species-specific cyclin T1 must be formed before the binding to TAR can occur with high affinity and specificity. In sharp contrast, Tat from the bovine immunodeficiency virus (BIV) binds to its TAR without the help of the cyclin T1. This binding depends on the upper stem and 5' bulge, but not the central loop in TAR. Moreover, cyclins T1 from different species can mediate effects of this Tat in cells. Unlike the situation with other lentiviruses, Tat transactivation can be rescued simply by linking a heterologous promoter to TAR in permissive cells. Thus, lentiviruses have evolved different strategies to recruit Tat and the positive transcription elongation factor b to their promoters, and interactions between Tat and TAR are independent from those between Tat and the cyclin T1 in BIV.

Animals↗
PubMed2004

Selection of TAR RNA-binding chameleon peptides by using a retroviral replication system.

The interaction between the arginine-rich motif (ARM) of the human immunodeficiency virus (HIV) Tat protein and TAR RNA is essential for Tat activation and viral replication. Two related lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), also require Tat ARM-TAR interactions to mediate activation, but the viruses have evolved different RNA-binding strategies. Interestingly, the JDV ARM can act as a "chameleon," adopting both the HIV and BIV TAR binding modes. To examine how RNA-protein interactions may evolve in a viral context and possibly to identify peptides that recognize HIV TAR in novel ways, we devised a retroviral system based on HIV replication to amplify and select for RNA binders. We constructed a combinatorial peptide library based on the BIV Tat ARM and identified peptides that, like the JDV Tat ARM, also function through HIV TAR, revealing unexpected sequence characteristics of an RNA-binding chameleon. The results suggest that a retroviral screening approach may help identify high-affinity TAR binders and may provide new insights into the evolution of RNA-protein interactions.

Amino Acid Sequence↗
PubMed2005

Inhibition of HIV derived lentiviral production by TAR RNA binding domain of TAT protein.

BACKGROUND: A critical step in the production of new HIV virions involves the TAT protein binding to the TAR element. The TAT protein contains in close proximity its TAR RNA binding domain and protein transduction domain (PTD). The PTD domain of TAT has been identified as being instrumental in the protein's ability to cross mammalian cell and nuclear membranes. All together, this information led us to form the hypothesis that a protein containing the TAR RNA binding domain could compete with the native full length TAT protein and effectively block the TAR RNA binding site in transduced HIV infected cells. RESULTS: We synthesized a short peptide named Tat-P, which contained the TAR RNA binding and PTD domains to examine whether the peptide has the potential of inhibiting TAT dependent HIV replication. We investigated the inhibiting effects of Tat-P in vitro using a HIV derived lentiviral vector model. We found that the TAT PTD domain not only efficiently transduced test cells, but also effectively inhibited the production of lentiviral particles in a TAT dependent manner. These results were also supported by data derived from the TAT activated LTR-luciferase expression model and RNA binding assays. CONCLUSION: Tat-P may become part of a category of anti-HIV drugs that competes with full length TAT proteins to inhibit HIV replication. In addition, this study indicates that the HIV derived lentiviral vector system is a safe and reliable screening method for anti-HIV drugs, especially for those targeting the interaction of TAT and TAR RNAs.

Anti-HIV Agents↗