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PubMed2002

Base flexibility in HIV-2 TAR RNA mapped by solution (15)N, (13)C NMR relaxation.

Binding of the HIV tat protein to the TAR (transactivating response region) RNA element activates transcription of the HIV viral genome. The complex of TAR with argininamide serves as a model for the RNA conformation in the tat-TAR complex. The dynamics of the HIV-2 TAR-argininamide complex was investigated by measurements of the relaxation rates of protonated base carbon and nitrogen nuclei. Six auto-correlation rates as well as cross-correlation rates were measured to map the frequencies of base motion in the HIV-2 TAR-argininamide complex. These measurements reveal a broad range of dynamic heterogeneity exhibited by hexanucleotide loop, the dinucleotide bulge, and the A-form helical regions. U23 in the bulge undergoes the largest dynamic change on binding argininamide, while U25 remains flexible, reflecting the large conformational change that is triggered upon ligand binding.

Arginine↗
PubMed1993

A chimeric human immunodeficiency virus type 1 TAR region which mediates high level trans-activation in both rodent and human cells.

Trans-activation of the HIV-1 LTR by the Tat protein functions by a novel mechanism which involves the direct interaction of the Tat protein and cellular factors with nascently transcribed viral RNA encoding the Tat responsive element (TAR). Rodent cells do not efficiently support HIV-1 Tat activity because of a deficiency of human-specific factor(s). Human chromosome 12 appears to encode one of these Tat cofactors. We have designed chimeric TAR sequences which contain the heterologous RNA sequence derived from the bacteriophage R17 genome which binds to the bacteriophage MS2 coat protein. These chimeric TAR constructs were co-transfected into rodent and human cells with a plasmid encoding a chimeric Tat protein which contains the RNA binding domain of the MS2 coat protein. TAR constructs which contain the MS2 coat protein binding region inserted into the three nucleotide "bulge" region support a high level of trans-activation by Tat-MS2 coat protein chimeras in both human and rodent cells. This result suggests that the human-specific Tat cofactor(s) may act to allow Tat to interact effectively in a ribonucleoprotein complex which includes Tat, cellular factors, and TAR RNA.

Animals↗
PubMed1996

Block of Tat-mediated transactivation of tumor necrosis factor beta gene expression by polymeric-TAR decoys.

The tat gene product (Tat) of human immunodeficiency virus type 1 (HIV-1) is an early regulatory protein which transactivates HIV-1 gene expression by interacting with the trans-activation response element (TAR) present in the HIV-1 long terminal repeat (LTR). In HIV-1-infected cells Tat can also activate the expression of tumor necrosis factor (TNF). Recent results indicate that essential for this effect is the interaction of Tat with a TAR-like structure present in the TNF beta messenger RNA leader region that closely resembles the TAR of the HIV-LTR. Here we show that because of this similarity of mechanisms, the expression of an RNA species encoding polymeric-TAR sequences and known to inhibit Tat-mediated HIV-1 gene expression also blocks TNF gene expression in response to Tat, but not TNF promoter activation induced by human T cell leukemia/lymphotropic virus type I Tax protein. Since TNF is increased in HIV-1-infected individuals and can activate HIV-1 gene expression or rescue Tat-defective HIV-1 proviruses, activation of TNF by Tat may be part of a complex pathway in which HIV-1 uses its own expression to increase infectivity and to induce disease. This study shows a dual role for the polymeric-TAR construct in inhibiting HIV-1 replication and strengthens the potential use of this protective gene in gene therapy for AIDS.

Gene Expression Regulation, Viral↗
PubMed1996

Structural and Functional Properties of HIV-1(GER) TAR Sequences.

Sequencing of HIV-1(GER) long terminal repeat (LTR) has demonstrated, for the first time in an HIV-1 primary isolate, a TAR duplication referred to as TAR1 (nucleotides +1 through +68) and TAR2 (nucleotides +69 through +136). This TAR duplication is stable during replication of HIV-1(GER) isolate in CEM cells. Analysis of LTR-CAT reporter constructs demonstrated that under Tat transactivation the HIV-1(GER)/LTR (containing TAR1 and TAR2) was expressed at a higher level than a similar construct (HIV-1(GER)DeltaTAR) containing a single TAR sequence. Among the two transcription initiation sites found in the HIV-1(GER)/LTR, only the most 5' start site was shown to be functionally active. The predicted secondary structure of the 5'-end mRNAs of HIV-1(GER) suggests it may fold into a double TAR hairpin which resembles that of HIV-2. Finally, HIV-1(GER) Tat protein shows primary sequence similarity with Tat proteins from other isolates of HIV-1 and is apparently unrelated to HIV-2 Tat proteins. This work provides the first evidence of a TAR sequence duplication in HIV-1 which increases the efficiency of transactivation by Tat. Copyright 1996 S. Karger AG, Basel

Journal Article↗
PubMed2001

Binding of phage-displayed HIV-1 Tat to TAR RNA in the presence of cyclin T1.

The transactivator protein (Tat) of the human immunodeficiency virus (HIV) is a key regulatory protein in the viral replication cycle. Together with cellular cyclin T1 and an RNA element (transactivation response; TAR) located at the 5' end of all viral transcripts, it forms a ternary complex that ultimately enhances the expression of all viral genes. In this ternary complex, cyclin T1 interacts directly with Tat and TAR. The presence of cyclin T1 is essential for high TAR RNA affinity and specificity of Tat. To study protein-protein and protein-RNA interaction, we developed a phage display system that displays functional Tat on the surface of bacteriophage M13. The addition of recombinant cyclin T1 to the selections yielded a phage display system that mirrors all binding properties of the cyclin T1-Tat-TAR complex known from cell assays and biochemical studies. Phage-displayed Tat protein as well as the cyclin T1 are fully functional. The relative binding capabilities of wild-type- and mutant Tat-displaying phages show that the presence of cyclin T1 significantly reduces the importance of basic residues in the basic sequence region of Tat for its binding to TAR.

Amino Acid Sequence↗
PubMed2006

Skin tumors among employees of a tar refinery: the current data and their implications.

OBJECTIVE: Skin tumors and their preliminary stages may be recognized and compensated as an occupational disease only after exposure to soot, raw paraffin, tar, anthracene, pitch or similar substances. The presented study was carried out in one of the world's largest tar refineries and special attention was paid to the dermatological long-term effects and today's relevance of tar-induced skin lesions. METHODS: The data were generated by means of a standardized questionnaire based on occupational disease records and dermatological reports that have been carried out on a regular basis retrospectively. Among other things, the localization, the number and type of skin tumor, the date of diagnosis and the histological evaluation, where available, were taken into account. RESULTS: In a longitudinal analysis from 1946 to 2002, a total of n = 618 employees were examined. Squamous cell carcinomas n = 393, basal cell carcinomas n = 298, keratoacanthomas n = 194 and melanomas n = 6 were diagnosed. Latency periods for keratoacanthomas (median 29.5 years) and squamous cell carcinomas (median 28.0 years) were almost identical and could possibly imply that the pathomechanisms for both types of skin tumor might be comparable. Of the persons diagnosed, the skin tumors were mainly localized in the head region (n = 1,662). A remarkable divergence in the localization and distribution of the lesions was found when compared to the general population, for example the relative susceptibility of the upper lip (n = 67) compared to the lower lip (n = 31) as well as the less sun-exposed forearms (n = 381) compared to the back of the hand (n = 331), being typically preferred sites of squamous cell carcinomas. Furthermore, a discrepancy between the distribution of premalignant nonmelanoma skin tumors and carcinomas on the back of the nose, which is more exposed to the sun (n = 56) and those in the nostrils (n = 89), which are less exposed to the sun, but are in the act of breathing more exposed to tar, could be found. CONCLUSIONS: Tar-induced skin lesions are still of importance nowadays, which is reflected in the incidence of new cases and in the relapses of skin tumors. As a consequence, intensified measures are necessary, in the form of primary and secondary prevention. In view of the high incidence of malignant skin tumors in the general public, it is urgently necessary to take occupational hazards into consideration as well.

Adult↗
PubMed1992

Trans-activation of H-1 parvovirus P38 promoter is correlated with increased binding of cellular protein(s) to the trans-activation responsive element (tar).

The parvovirus H-1 P38 promoter contains a trans-activation responsive element (tar). It was previously shown that the parvovirus H-1 nonstructural protein NS1 positively regulates the expression of the P38 promoter for the viral capsid protein gene via the tar (Rhode and Richard, 1987, J. Virol. 61, 2807-2515). To characterize the mechanism of trans-activation by the tar, we used gel shift assays to demonstrate that there exist proteins in virus-infected cellular extracts which have higher binding activity than that found in mock-infected extracts. These observations in vitro are consistent with the expression by P38 constructs with the wild-type promoter linked to a reporter gene, chloramphenicol acetyl transferase (cat), in vivo. We also provide evidence that the protein(s)-tar complex has a molecular mass of approximately 75 kDa in an SDS-polyacrylamide gel, which is less than NS1, and this complex cannot be precipitated by NS1 antibody, which suggests that NS1 mediates the trans-activation by inducing an alteration in the binding activity of some cellular protein(s) in an indirect manner. These data support our previous hypothesis for the activation of the P38 promoter, in which the trans-activator(s) interacts with the tar effectively in the presence of NS1, leading to the formation of the transcription initiation complex by protein-protein associations (Gu, Chen, and Rhode, 1992, Virology 187, 10-17).

Base Sequence↗
PubMed1989

Activation of double-stranded RNA-dependent kinase (dsl) by the TAR region of HIV-1 mRNA: a novel translational control mechanism.

All mRNAs of human immunodeficiency virus 1 (HIV-1) contain in their 5' untranslated region a sequence termed TAR that responds to trans-activation by the tat (trans-activating) protein. This RNA sequence assumes a stable secondary structure, and its cap structure is relatively inaccessible. Here we report that these structural properties of the TAR sequence underlie the ability of TAR to inhibit in trans the translation of other mRNAs. This mechanism of translation inhibition involves the activation of the double-stranded RNA-dependent kinase (dsl), which in turn phosphorylates the protein synthesis initiation factor 2 (eIF-2). Mutations in the TAR region that diminish the stability of the secondary structure cause a significant reduction in the trans-inhibition. A similar reduction in the dsl activation occurs when TAR is placed further downstream of the cap structure. This is a clear demonstration of a specific naturally occurring mRNA sequence that can activate dsl. We suggest a novel translational regulatory mechanism that interdigitates the activities of eIF-2 and eIF-4F.

Animals↗
PubMed1990

A nuclear translational block imposed by the HIV-1 U3 region is relieved by the Tat-TAR interaction.

Replication of HIV-1 depends on the viral Tat protein, which functions via a target sequence, TAR, present in the proviral long terminal repeat (LTR) and at the 5' end of viral mRNAs. We have shown that Tat potentiates the expression of TAR-containing RNAs, but only when Tat and the TAR-containing RNA are present in the nucleus. We now show that a small change in the TAR loop abolishes nuclear potentiation by Tat. Furthermore, the HIV-1 U3 region induces expression incompetence in mRNA synthesized by this promoter. RNAs of identical structure are, however, translated efficiently when produced from the CMV-IE promoter. The Tat-TAR system appears, therefore, to rescue the expression potential of HIV-1 LTR-directed RNA.

Animals↗
PubMed1989

Plastic particle and tar pollution on beaches of Kuwait.

Twelve beaches on the northwestern Arabian Gulf coast of Kuwait were surveyed for the occurrence of plastic particles, tar balls and tar lumps. Particles were found on all beaches and were abundant on four: Suleibkhat, Fintas, Fahaheel and New Al Khiran. Most particles were composed of low-density polyethylene, but a few were of high-density polyethylene, polypropylene and polystyrene. Many were weathered and had traces of tar. Tar lumps and balls were present on all beaches, but were not as abundant as was expected, considering the heavy volume of oil tanker traffic in this region of the Arabian Gulf. Tar appeared to be less abundant than it was in 1979. With at least 33 plastics factories operating on the coast of Kuwait, it is possible that plastic particles occur more abundantly than has been observed in this study. The high winds and concurrent wave activity, which often vary in strength and direction but regularly occur here, probably play an important role in dispersing plastic particles which are washed up on to these beaches, making it difficult to accurately measure their abundance.

Journal Article↗
PubMed2003

Quantitative study of HIV-1 Tat peptide and TAR RNA interaction inhibited by poly(allylamine hydrochloride).

The interaction of poly(allylamine hydrochloride) (PAH) with TAR RNA has been studied by quartz crystal microbalance (QCM) cooperating with capillary electrophoresis (CE). Experimental results showed that PAH had high affinity for TAR RNA. In particular, PAH could disrupt the interaction of Tat peptide with TAR RNA, which is critical for HIV-1 virus replication. The approaches described here indicate that they are powerful for studying the binding processes of Tat peptide-TAR RNA and drug-TAR RNA, having great significance for the design of new drug.

Biosensing Techniques↗
PubMed2004

Alpha,alpha-trehalose derivatives bearing guanidino groups as inhibitors to HIV-1 Tat-TAR RNA interaction in human cells.

Replication of HIV-1 requires specific interactions of Tat protein with TAR RNA. Disruption of Tat-TAR RNA interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for blocking the interaction of Tat-TAR RNA. The core molecule 6,6'-diamino-6,6'-dideoxy-alpha,alpha-trehalose was obtained from selective bromination of, alpha,alpha-trehalose at C-6,6', followed by acetylation, azide displacement, deacetylation, and reduction. Coupling of the core molecule with the protected amino acid, then deprotection and guanidinylation generated the novel alpha,alpha-trehalose derivatives. Their abilities to inhibit Tat-TAR RNA interaction in human cells were determined by a Tat-dependent HIV-1 LTR-driven CAT assays.

Anti-HIV Agents↗
PubMed2006

Specific recognition of HIV TAR RNA by the dsRNA binding domains (dsRBD1-dsRBD2) of PKR.

PKR (double-stranded RNA-dependent protein kinase) is an important component of host defense to virus infection. Binding of dsRNA to two dsRBDs (double-stranded RNA binding domains) of PKR modulates its own kinase activation. How structural features of natural target RNAs, such as bulges and loops, have an effect on the binding to two dsRBDs of PKR still remains unclear. By using ITC and NMR, we show here that both the bulge and loop of TAR RNA are necessary for the high affinity binding to dsRBD1-dsRBD2 of PKR with 1:1 stoichiometry. The binding site for the dsRBD1-dsRBD2 spans from upper bulge to lower stem of the TAR RNA, based on chemical shift mapping. The backbone resonances in the 40 kDa TAR.dsRBD1-dsRBD2 were assigned. NMR chemical shift perturbation data suggest that the beta1-beta2 loop of the dsRBD1 interacts with the TAR RNA, whereas that of the dsRBD2 is less involved in the TAR RNA recognition. In addition, the residues of the interdomain linker between the dsRBD1 and the dsRBD2 also show large chemical perturbations indicating that the linker is involved in the recognition of TAR RNA. The results presented here provide the biophysical and spectroscopic basis for high-resolution structural studies, and show how local RNA structural features modulate recognition by dsRBDs.

Amino Acid Sequence↗
PubMed2004

Evidence for conformational flexibility in the Tat-TAR recognition motif of cyclin T1.

Cyclin T1 (CycT1) is a cellular transcription elongation factor that also participates in Tat-mediated activation of several lentiviral promoters. In human immunodeficiency virus (HIV), CycT1 is required for Tat to bind tightly to TAR and interacts in the ternary complex via its Tat-TAR recognition motif (TRM). In the related bovine immunodeficiency virus (BIV), Tat recognizes its cognate TAR element with high affinity and specificity in the absence of CycT1. At both promoters, CycT1 recruits the Cdk9 kinase, which phosphorylates RNA polymerase II to generate processive transcription complexes. To examine the physical properties of CycT1, we purified a functional domain corresponding to residues 1-272 and found that it possesses a stably folded core, as judged by partial proteolysis and circular dichroism experiments. Interestingly, the C-terminal 20 residues corresponding to the TRM appear conformationally flexible or disordered. The TRM of the bovine CycT1 (bCycT1) is similarly sensitive to proteolysis yet differs in sequence from the human protein. In particular, bCycT1 lacks a cysteine at residue 261 known to be critical for HIV but not BIV ternary complex formation, and mutagenesis data are consistent with a proposed role for this cysteine in metal binding. The apparent flexibility of the TRM suggests that conformational rearrangements may accompany formation of CycT1-Tat-TAR ternary complexes and may contribute to different TAR recognition strategies in different lentiviruses.

Amino Acid Sequence↗
PubMed1997

Specific inhibition of in vitro reverse transcription using antisense oligonucleotides targeted to the TAR regions of HIV-1 and HIV-2.

Antisense oligonucleotides (ODNs) overlapping the stem-loop structure of the trans-activating responsive (TAR) element at the 5' end of HIV-1 and HIV-2 viral RNAs were tested for their inhibitory effect on cDNA synthesis by HIV-1 and HIV-2 reverse transcriptases (RT). Inhibition of reverse transcription is sequence-specific and enhanced by the presence of the RT-associated RNase H activity. The degree of inhibition obtained with the anti-TAR antisense is significantly higher than with other HIV-1 targeted antisense ODNs used before [1]. Gel retardation showed a stable specific complex between the 16- and 25-mer anti-TAR HIV-1 selected ODNs and the target region. No complex was observed with a non-inhibitor 22-mer anti-TAR ODN and with the corresponding control sequences. Targeting of the first stem-loop in the 5' region of HIV-2 RNA by anti-TAR ODNs inhibited very strongly reverse transcription by HIV-2 RT. The structure of the antisense and the target sequence affect annealing efficiency and hence the degree of inhibition of reverse transcription.

Base 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↗
PubMed2001

Interaction of new PNA-based molecules with TAR RNA of HIV-1: molecular modelling and biological evaluation.

During the HIV-1 replication process, interactions between the RNA sequence, named TAR RNA, and the viral protein, Tat, permit a fast and efficient transcription of viral DNA into RNA. Based on the NMR structure of TAR RNA from the PDB, two Peptidic Nucleic Analog- (PNA) based molecules were designed by molecular modelling, the first one targeting G32 U31 and the second targeting U31 C30 free loop bases. Before designing the molecules, the flexibility of the TAR RNA was evaluated by molecular dynamics (MD). The molecules studied are composed of three domains: an arginine, a linker, and two PNA bases. First, molecules were designed and the linker length was optimized to fit the TAR RNA; second, a MD simulation on the TAR RNA molecule complex was performed to validate the molecular structure. Optimal molecules were synthesized and tested on infected cells. The experimental results support the choices made in the design of the molecules.

Circular Dichroism↗
PubMed2003

Inhibition of HIV-1 gene expression by novel DNA enzymes targeted to cleave HIV-1 TAR RNA: potential effectiveness against all HIV-1 isolates.

Nucleic acid-based antiviral approaches have been tried against multiple HIV-1 genes with the purpose of down-regulating its replication. A unique stem-loop structure called TAR is present at the 5'-end of all HIV-1 transcripts; Tat and other cellular proteins bind to TAR and thus govern transcription. Therefore, HIV-1 TAR is an attractive target against which various antiviral approaches could be tried. We screened several DNA enzymes (Dz's) containing the 10-23 catalytic motif and a single Dz containing the 8-17 catalytic motif against the HIV-1 TAR RNA. Dz's directed against the predicted single-stranded bulge regions showed sequence-specific cleavage activities. One of the two Dz's, namely Dz-475, showed moderate cleavage activity in complete absence of Mg(2+). Addition of unrelated sequences at the 5'-end of the HIV-1 TAR RNA rendered it susceptible to four additional Dz-mediated cleavages. Both Dz's (470 and 475) showed significant intracellular reduction of HIV-1 gene expression. Dz-475-treated cells showed significant protection against T-tropic and macrophage-tropic HIV-1 challenge. Dz-475-transfected T-lymphocytes, human PBMCs, or chronically infected cell lines showed marked viral resistance. Unique features of this antiviral strategy with respect to HIV-1 gene inhibition are discussed.

Anti-HIV Agents↗