Trans-activation Responsive Region RNA Research Articles

Design-Based Peptidomimetic Ligand Discovery to Target HIV TAR RNA Using Comparative Analysis of Different Docking Methods

Discovering molecules capable of binding to HIV trans-activation responsive region (TAR) RNA thereby disrupting its interaction with Tat protein is an attractive strategy for developing novel antiviral drugs. Computational docking is considered as a useful tool for predicting binding affinity and conducting virtual screening. Although great progress in predicting protein-ligand interactions has been achieved in the past few decades, modeling RNA-ligand interactions is still largely unexplored due to the highly flexible nature of RNA. In this work, we performed molecular docking study with HIV TAR RNA using previously identified cyclic peptide L22 and its analogues with varying affinities toward HIV-1 TAR RNA. Furthermore, sarcosine scan was conducted to generate derivatives of CGP64222, a peptide-peptoid hybrid with inhibitory activity on Tat/TAR RNA interaction. Each compound was docked using CDOCKER, Surflex-Dock and FlexiDock to compare the effectiveness of each method. It was found that FlexiDock energy values correlated well with the experimental Kd values and could be used to predict the affinity of the ligands toward HIV-1 TAR RNA with a superior accuracy. Our results based on comparative analysis of different docking methods in RNA-ligand modeling will facilitate the structure-based discovery of HIV TAR RNA ligands for antiviral therapy.

Probing interaction of a fluorescent ligand with HIV TAR RNA

Trans-activator of Transcription (Tat) antagonists could block the interaction between Tat protein and its target, trans-activation responsive region (TAR) RNA, to inhibit Tat function and prevent human immunodeficiency virus type 1 (HIV-1) replication. For the first time, a small fluorescence ligand, ICR 191, was found to interact with TAR RNA at the Tat binding site and compete with Tat. It was also observed that the fluorescence of ICR 191 could be quenched when binding to TAR RNA and recovered when discharged via competition with Tat peptide or a well-known Tat inhibitor, neomycin B. The binding parameters of ICR 191 to TAR RNA were determined through theoretical calculations. Mass spectrometry, circular dichroism and molecular docking were used to further confirm the interaction of ICR 191 with TAR RNA. Inspired by these discoveries, a primary fluorescence model for the discovery of Tat antagonists was built using ICR 191 as a fluorescence indicator and the feasibility of this model was evaluated. This ligand-RNA interaction could provide a new strategy for research aimed at discovering Tat antagonists.

Iron(II) supramolecular helicates interfere with the HIV-1 Tat-TAR RNA interaction critical for viral replication.

The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat–TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

The N-Terminal Zinc Finger and Flanking Basic Domains Represent the Minimal Region of the Human Immunodeficiency Virus Type-1 Nucleocapsid Protein for Targeting Chaperone Function

The human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein is a chaperone that facilitates nucleic acid conformational changes to produce the most thermodynamically stable arrangement. The critical role of NC in many steps of the viral life cycle makes it an attractive therapeutic target. The chaperone activity of NC depends on its nucleic acid aggregating ability, duplex destabilizing activity, and rapid on-off binding kinetics. During the minus-strand transfer step of reverse transcription, NC chaperones the annealing of highly structured transactivation response region (TAR) RNA to the complementary TAR DNA. In this work, the role of different functional domains of NC in facilitating 59-nucleotide TAR RNA-DNA annealing was probed by using chemically synthesized peptides derived from full-length (55 amino acids) HIV-1 NC: NC(1-14), NC(15-35), NC(1-28), NC(1-35), NC(29-55), NC(36-55), and NC(11-55). Most of these peptides displayed significantly reduced annealing kinetics, even when present at concentrations much higher than that of wild-type (WT) NC. In addition, these truncated NC constructs generally bind more weakly to single-stranded DNA and are less effective nucleic acid aggregating agents than full-length NC, consistent with the loss of both electrostatic and hydrophobic contacts. However, NC(1-35) displayed annealing kinetics, nucleic acid binding, and aggregation activity that were very similar to those of WT NC. Thus, we conclude that the N-terminal zinc finger, flanked by the N-terminus and linker domains, represents the minimal sequence that is necessary and sufficient for chaperone function in vitro. In addition, covalent continuity of the 35 N-terminal amino acids of NC is critical for full activity. Thus, although the hydrophobic pocket formed by residues proximal to the C-terminal zinc finger has been a major focus of recent anti-NC therapeutic strategies, NC(1-35) represents an alternative target for therapeutics aimed at disrupting NC's chaperone function.

Interactions of protein side chains with RNA defined with REDOR solid state NMR

Formation of the complex between human immunodeficiency virus type-1 Tat protein and the transactivation response region (TAR) RNA is vital for transcriptional elongation, yet the structure of the Tat-TAR complex remains to be established. The NMR structures of free TAR, and TAR bound to Tat-derived peptides have been obtained by solution NMR, but only a small number of intermolecular NOEs could be identified unambiguously, preventing the determination of a complete structure. Here we show that a combination of multiple solid state NMR REDOR experiments can be used to obtain multiple distance constraints from (15)N to (13)C spins within the backbone and side chain guanidinium groups of arginine in a Tat-derived peptide, using (19)F spins incorporated into the base of U23 in TAR and (31)P spins in the P22 and P23 phosphate groups. Distances between the side chain of Arg52 and the base and phosphodiester backbone near U23 measured by REDOR NMR are comparable to distances observed in solution NMR-derived structural models, indicating that interactions of TAR RNA with key amino acid side chains in Tat are the same in the amorphous solid state as in solution. This method is generally applicable to other protein-RNA complexes where crystallization or solution NMR has failed to provide high resolution structural information.

Synthesis and Single-Molecule Characterization of a DNA Hairpin Construct Based on the TAR RNA Sequence

Optical tweezers are used to examine the folding and unfolding of single molecules, revealing the effects of force, ionic strength, proteins, and small molecules on the kinetic and thermodynamic parameters of bimolecular stability. Here, we examine a DNA hairpin construct related to the trans-activation responsive region (TAR) RNA sequence of HIV-1. In an optical tweezers experiment, a bead at one end of a duplex DNA assembly is attached to a micropipette, while a bead at the other end is held in an optical trap, thus allowing investigators to relate picoNewton forces necessary to unfold the DNA with corresponding nanometer changes in length. Force extension/relaxation data are used to elucidate DNA folding and unfolding processes. Our DNA construct design contains a central hairpin structure with long double-stranded “handles” attached to both the 3’ and 5’ end of the hairpin. The DNA “handles”, each approximately 3 kb in length, were synthesized by PCR using biotin- and digoxigenin-labeled primers that serve to attach the final construct to 5 µm-diameter beads for use with the optical tweezers. The smaller central hairpin sequence was obtained via solid-phase organic synthesis. After purification and assembly with a linker oligonucleotide, the 5’ handle and the hairpin structure were ligated together and purified by agarose gel electrophoresis. This product was then ligated to the 3’ handle and again purified. Individual molecules of the final hairpin construct, approximately 6 kb in length, were bound to streptavidin or anti-digoxigenin-labeled beads and their unfolding behavior was studied using optical tweezers.

HIV-1 TAR RNA Spontaneously Undergoes Relevant Apo-to-Holo Conformational Transitions in Molecular Dynamics and Constrained Geometrical Simulations

We report all-atom molecular dynamics and replica exchange molecular dynamics simulations on the unbound human immunodeficiency virus type-1 (HIV-1) transactivation responsive region (TAR) RNA structure and three TAR RNA structures in bound conformations of, in total, approximately 250 ns length. We compare the extent of observed conformational sampling with that of the conceptually simpler and computationally much cheaper constrained geometrical simulation approach framework rigidity optimized dynamic algorithm (FRODA). Atomic fluctuations obtained by replica-exchange molecular dynamics (REMD) simulations agree quantitatively with those obtained by molecular dynamics (MD) and FRODA simulations for the unbound TAR structure. Regarding the stereochemical quality of the generated conformations, backbone torsion angles and puckering modes of the sugar-phosphate backbone were reproduced equally well by MD and REMD simulations, but further improvement is needed in the case of FRODA simulations. Essential dynamics analysis reveals that all three simulation approaches show a tendency to sample bound conformations when starting from the unbound TAR structure, with MD and REMD simulations being superior with respect to FRODA. These results are consistent with the experimental view that bound TAR RNA conformations are transiently sampled in the free ensemble, following a conformation selection model. The simulation-generated TAR RNA conformations have been successfully used as receptor structures for docking. This finding has important implications for RNA-ligand docking in that docking into an ensemble of simulation-generated RNA structures is shown to be a valuable means to cope with large apo-to-holo conformational transitions of the receptor structure.

RNA Dimerization Promotes PKR Dimerization and Activation

The double-stranded RNA (dsRNA)-activated protein kinase [protein kinase R (PKR)] plays a major role in the innate immune response in humans. PKR binds dsRNA non-sequence specifically and requires a minimum of 15-bp dsRNA for one protein to bind and 30-bp dsRNA to induce protein dimerization and activation by autophosphorylation. PKR phosphorylates eukaryotic initiation factor 2α, a translation initiation factor, resulting in the inhibition of protein synthesis. We investigated the mechanism of PKR activation by an RNA hairpin with a number of base pairs intermediate between these 15- to 30-bp limits: human immunodeficiency virus type 1 transactivation-responsive region (TAR) RNA, a 23-bp hairpin with three bulges that is known to dimerize. TAR monomers and dimers were isolated from native gels and assayed for RNA and protein dimerization to test whether RNA dimerization affects PKR dimerization and activation. To modulate the extent of dimerization, we included TAR mutants with different secondary features. Native gel mixing experiments and analytical ultracentrifugation indicate that TAR monomers bind one PKR monomer and that TAR dimers bind two or three PKRs, demonstrating that RNA dimerization drives the binding of multiple PKR molecules. Consistent with functional dimerization of PKR, TAR dimers activated PKR while TAR monomers did not, and RNA dimers with fewer asymmetrical secondary-structure defects, as determined by enzymatic structure mapping, were more potent activators. Thus, the secondary-structure defects in the TAR RNA stem function as antideterminants to PKR binding and activation. Our studies support that dimerization of a 15- to 30-bp hairpin RNA, which effectively doubles its length, is a key step in driving activation of PKR and provide a model for how RNA folding can be related to human disease.

Single-Molecule Study of the Inhibition of HIV-1 Transactivation Response Region DNA/DNA Annealing by Argininamide

Single-molecule spectroscopy was used to examine how a model inhibitor of HIV-1, argininamide, modulates the nucleic acid chaperone activity of the nucleocapsid protein (NC) in the minus-strand transfer step of HIV-1 reverse transcription, in vitro. In minus-strand transfer, the transactivation response region (TAR) RNA of the genome is annealed to the complementary "TAR DNA" generated during minus-strand strong-stop DNA synthesis. Argininamide and its analogs are known to bind to the hairpin bulge region of TAR RNA as well as to various DNA loop structures, but its ability to inhibit the strand transfer process has only been implied. Here, we explore how argininamide modulates the annealing kinetics and secondary structure of TAR DNA. The studies reveal that the argininamide inhibitory mechanism involves a shift of the secondary structure of TAR, away from the NC-induced "Y" form, an intermediate in reverse transcription, and toward the free closed or "C" form. In addition, more potent inhibition of the loop-mediated annealing pathway than stem-mediated annealing is observed. Taken together, these data suggest a molecular mechanism wherein argininamide inhibits NC-facilitated TAR RNA/DNA annealing in vitro by interfering with the formation of key annealing intermediates.

Isothermal Calorimetric Studies of Arginine and Divalent Ion Binding to the TAR RNA

The transactivation-response region (TAR) of HIV-1 increases its rate of transcription by one-hundred fold upon binding to the Tat protein. The TAR RNA of HIV-1 contains a conserved three nucleotide bulge: U23, C23, and U25; the crystal structure shows three calcium ions bound to the bulge. The base-triple, U23·A27-U38, is important for arginine binding (R52 of Tat). Thermal denaturation experiments on TAR RNA show that binding of divalent metal ions increases its stability relative to 1 M K+ ions. Isothermal Titration Calorimetry was used to measure the binding constant and enthalpy of binding for arginine and divalent metal ions. Arginine binds at one site on the TAR RNA whereas calcium and magnesium bind at a minimum of two sites. Calcium and magnesiumions also have different binding affinities for the two sites.

Orientation and Affinity of HIV-1 Tat Fragments in Tat−TAR Complex Determined by Fluorescence Resonance Energy Transfer

The human immunodeficiency virus (HIV-1) encodes a transcriptional activator protein, Tat, which is expressed early in the viral life cycle and is essential for viral gene expression, replication, and pathogenesis. Tat interacts with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. Tat-derived peptides that contain the basic arginine-rich region of Tat are able to form in vitro complexes with TAR RNA, and these peptides provide a well-characterized system for understanding the mechanism of RNA-protein recognition. It is not known how RNA-binding Tat peptides are folded or docked in the Tat-TAR complex, and to what extent structural reorganization occurs upon TAR binding. To address these questions, we developed a fluorescence resonance energy transfer (FRET) system to analyze the interactions between TAR RNA and a Tat protein fragment (aa 38-72) uniquely labeled with donor and acceptor dye molecules, respectively. Using this FRET assay, we determined the binding affinity of Tat (47-58) and Tat (38-72) for TAR RNA under physiological conditions. We also delineated the distance between the N- and C-terminus of Tat (38-72) and the distance between the two termini and the 5' end of TAR when Tat (38-72) is bound to TAR. Our results suggest that the N- and C-termini of Tat (38-72) are close to each other when the peptide is folded and that the peptide does not go through a large structural change upon TAR binding.

Novel Second Generation Anti-HIV shRNA Expressing vif and Decoy TAR Arrest the Virus-breakthrough Phenomenon Associated With siRNA-escape Variants

We describe a novel chimera RNA expressing vif shorthairpin RNA (shRNA) and decoy trans-activation response region (TAR) RNA from a human U6 Pol II promoter, which enhanced the inhibition of human immunodeficiency virus (HIV) vif small-interfering RNA (siRNA) and arrested virus breakthrough by siRNA-generated escape variants in long-term culture assays. Our strategy was based on a second-generation anti-HIV-1 shRNA vector system, in which HIV-1 vif shRNA was fused to a decoy TAR RNA by a linker UU cleavage site to generate vif shRNA-decoy TAR RNA. Upon expression, the RNA molecule was cleaved and separated into vif siRNA and decoy TAR RNA. The synergistic effect of these molecules enhanced the inhibition of HIV-1 replication in a longterm culture assay and prevented virus breakthrough associated with siRNA-mediated escape variants. Combining shRNA with decoy TAR RNA as second-generation antiHIV shRNA may provide practical basis for applying siRNA-based gene therapy to the treatment of HIV/AIDS. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Discoveries of Tat-TAR Interaction Inhibitors for HIV-1

A major problem associated with anti-HIV-1 treatment is rapid emergence of drug-resistant strains. Accordingly, a compelling need is to discover anti-HIV drugs against alternative viral targets in addition to HIV-1 RT, PR, IN and CCR5. One such target is the interaction between HIV Trans-activator of transcription (Tat) protein and Trans Activation Responsive region (TAR) RNA. An arginine-rich motif (ARM) of Tat recognizing both the base sequence and the active conformation of TAR RNA three-base bulge region as well as newly elucidated TAR RNA inactive conformation are important for the specific Tat-TAR interaction. According to the possible binding modes, the inhibitors have been mainly divided into two classes: (1) Compounds binding directly to TAR RNA either to the TAR RNA three-base bulge region alone or to the three-base bulge together with the lower and upper-stem/Loop region. (2) Compounds binding directly to Tat protein with high affinity, thus potently inhibiting HIV-1. They both block Tat trans-activation in the formation of the Tat/TAR complex to exert antiviral activity in primary human cells. Recent researches also focus on the drugs targeting specificity of Tat and TAR by such new assays as capillary electrophoresis and quartz crystal microbalance. Cell-based reporter systems are established for high-throughput screening of novel compounds that interfere with Tat transactivation. The identification of dominant-negative mutants also finds wide application in this field. The Tat-TAR interaction is an important target in efforts to develop anti-HIV gene therapy or potential therapeutic antiviral agents for the treatment of HIV-1 infections.

Capillary Zone Electrophoretic Separation of Four Novel Compounds Active Against Human Immunodeficiency Virus Type 1

The separation of four novel small molecules active in the treatment of acquired immunodeficiency syndrome (AIDS), is presented. The molecules were synthesized by computer-aided design with the property of inhibiting the binding of the human immunodeficiency virus type 1 trans-activator of transcription (HIV-1 Tat) protein to the trans-activation response region (TAR) RNA required for HIV-1 transcription, and thus blocking of the HIV replication cycle. The active compounds were separated to baseline by capillary zone electrophoresis (CZE) under optimized conditions with 0.1 mol L−1 ammonium acetate solution (pH 3.8) as running buffer and UV detection. The method will be used study the interactions between the anti-HIV-1 active molecules and HIV TAR RNA and for rapid screening of these potential drugs for early drug discovery.

Inhibition of HIV-1 TAR RNA–Tat peptide complexation using poly(acrylic acid)

HIV-1 is regulated at the transcriptional level by the interaction of Tat protein with the transactivation responsive region (TAR) RNA, a 59-base stem–loop structure located at the 5 ′-end of all nascent HIV-1 transcripts. Here, by targeting the Tat peptide, we found that negatively charged poly(acrylic acid) (PAA) had high affinity with Tat peptide and could inhibit the interaction of TAR with Tat. Therefore, PAA could block HIV replication by binding to Tat not to TAR RNA, providing a new thinking for the design of novel anti-HIV drugs.

Design and Synthesis of Guanidinoglycosides Directed against the TAR RNA of HIV‐1

AbstractReplication of human immunodeficiency virus type 1 (HIV‐1) requires specific interactions of the Tat protein with the transactivation responsive region (TAR) RNA. Tat‐TAR RNA Interaction is mediated by a short arginine‐rich domain of the protein. Disruption of this interaction could, in theory, create a state of complete viral latency. Here, four novel 6‐amino‐6‐deoxytrehalose guanidinoglycoside derivatives (10 and 13–15) as target molecules have been designed to bind to TAR RNA for blocking the interaction of Tat‐TAR RNA. They were obtained by coupling 6‐amino‐6‐deoxy‐α,α‐trehalose (6) with the protected amino acids, deprotection by catalytic hydrogenation, followed by guanidinylated with S‐methylisothiourea sulfate. Their abilities to inhibit Tat‐TAR RNA interaction were determined by a Tat‐dependent HIV‐1 long terminal repeats (LTR)‐driven chloramphenicol acetyltransferase (CAT) assays.

Binding affinity and inhibitory potency of neomycin and streptomycin on the Tat peptide interaction with HIV-1 TAR RNA detected by on-line acoustic wave sensor.

The binding of two aminoglycoside antibiotics, neomycin and streptomycin, to a segment of the transactivation responsive region (TAR) RNA of the human immunodeficiency virus, and their inhibitory potency to disrupt the interaction of the RNA with a regulatory Tat protein-derived peptide, have been studied using a flow-through acoustic wave detector system. Binding affinity is directly correlated with the inhibitory potency of these molecules and the acoustic wave detection system shows that neomycin exhibits at least a ten-fold greater affinity for TAR RNA and that it is also a more potent inhibitor than streptomycin. These results are in agreement with previous studies. However, unlike the time-consuming batch-based assays, use of the flow-through format offers considerable potential for the rapid screening of the chemistry of relatively small-molecule-nucleic acid binding events.

A new class of RNA-binding oligomers: peptoid amide and ester analogues.

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. Here we report the design, synthesis and in vitro activities of oligopeptoid amide and ester analogues which bind TAR RNA with high affinities. These results show that we have identified a new class of unnatural oligomers for RNA targeting.

TAR RNA loop: a scaffold for the assembly of a regulatory switch in HIV replication.

Replication of HIV requires the Tat protein, which activates elongation of RNA polymerase II transcription at the HIV-1 promoter by interacting with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex b (P-TEFb). The transactivation domain of Tat binds directly to the CycT1 subunit of P-TEFb and induces loop sequence-specific binding of P-TEFb onto nascent HIV-1 trans-activation responsive region (TAR) RNA. We used systematic RNA-protein photocross-linking, Western blot analysis, and protein footprinting to show that residues 252-260 of CycT1 interact with one side of the TAR RNA loop and enhance interaction of Tat residue K50 to the other side of the loop. Our results show that TAR RNA provides a scaffold for two protein partners to bind and assemble a regulatory switch in HIV replication. RNA-mediated assembly of RNA-protein complexes could be a general mechanism for stable ribonucleoprotein complex formation and a key step in regulating other cellular processes and viral replication.

Tat Acetyl-acceptor Lysines Are Important for Human Immunodeficiency Virus Type-1 Replication

The human immunodeficiency virus type-1 trans-activator Tat is a transcription factor that activates the HIV-1 promoter through binding to the trans-activation-responsive region (TAR) localized at the 5'-end of all viral transcripts. We and others have recently shown that Tat is directly acetylated at lysine 28, within the activation domain, and lysine 50, in the TAR RNA binding domain, by Tat-associated histone acetyltransferases p300, p300/CBP-associating factor, and hGCN5. Here, we show that mutation of acetyl-acceptor lysines to arginine or glutamine affects virus replication. Interestingly, mutation of lysine 28 and lysine 50 differentially affected Tat trans-activation of integrated versus nonintegrated long terminal repeat. Our results highlight the importance of lysine 28 and lysine 50 of Tat in virus replication and Tat-mediated trans-activation.