Evidence for a base triple in the free HIV-1 TAR RNA

We propose the existence of a novel base triple in the HIV-1 TAR hairpin. This triple is supported by covariation of loop residue 31 with residue 22, which is part of an unusual base pair with U40 below the 3-nucleotide bulge. A set of mutants was constructed to test the involvement of bases A22, U31, and U40 in a triple interaction. RNA structure probing, trans-activation assays, and structure modeling are consistent with the existence of this base triple in a bent conformation of the free TAR element. However, disruption of the base triple does not affect binding of a Tat-derived peptide. We therefore compared the structure of free and Tat-bound TAR RNA by footprinting and site-specific cross-linking analyses. These studies indicate that the Tat arginine-rich motif, in addition to its known binding site at the bulge, is in close contact with U31 in the TAR loop. Because binding of Tat to TAR is known to coincide with the formation of a base triple with residues U23, A27, and U38, we hypothesize that Tat binding and the associated straightening of TAR triggers the disruption of the (A22-U40)U31 triple.     

Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure.

Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat.     

RNA recognition by Tat-derived peptides: interaction in the major groove?

Replication of human immunodeficiency virus requires binding of the viral Tat protein to its RNA target sequence TAR; peptides derived from Tat bind to a TAR "contact site" spanning 5 bp and a trinucleotide pyrimidine bulge. We find that high affinity binding requires a U residue in the bulge loop and 2 specific adjacent base pairs. Other bulged RNAs bind in a lower affinity nonspecific manner; sequence-specific binding requires a bulge loop of more than 1 nucleotide. Reaction with diethyl pyrocarbonate indicates that one effect of the bulge is to make the otherwise deep and narrow RNA major groove accessible. A model consistent with these data involves local distortion of A-form geometry at the bulge, which bends the helix and permits protein binding and interactive access in the RNA major groove.     

Argininamide binding arrests global motions in HIV-1 TAR RNA: comparison with Mg2+-induced conformational stabilization

The structure and dynamics of the stem-loop transactivation response element (TAR) RNA from the human immunodeficiency virus type-1 (HIV-1) bound to the ligand argininamide (ARG) has been characterized using a combination of a large number of residual dipolar couplings (RDCs) and trans-hydrogen bond NMR methodology. Binding of ARG to TAR changes the average inter-helical angle between the two stems from approximately 47 degrees in the free state to approximately 11 degrees in the bound state, and leads to the arrest of large amplitude (+/-46 degrees ) inter-helical motions observed previously in the free state. While the global structural dynamics of TAR-ARG is similar to that previously reported for TAR bound to Mg2+, there are substantial differences in the hydrogen bond alignment of bulge and neighboring residues. Based on a novel H5(C5)NN experiment for probing hydrogen-mediated 2hJ(N,N) scalar couplings as well as measured RDCs, the TAR-ARG complex is stabilized by a U38-A27.U23 base-triple involving an A27.U23 reverse Hoogsteen hydrogen bond alignment as well as by a A22-U40 Watson-Crick base-pair at the junction of stem I. These hydrogen bond alignments are not observed in either the free or Mg2+ bound forms of TAR. The combined conformational analysis of TAR under three states reveals that ligands and divalent ions can stabilize similar RNA global conformations through distinct interactions involving different hydrogen bond alignments in the RNA.     

Structural study of an RNA aptamer for a Tat protein complexed with ligands.

An RNA aptamer for an HIV Tat protein has been isolated by the in vitro SELEX method. The RNA aptamer binds to the Tat protein 50-100 times more strongly than native TAR RNA does. Here, we have investigated the structure of the RNA aptamer complexed with ligands, partial peptide fragments of the Tat protein or argininamide, by multidimensional 1H/13C/15N NMR. It is strongly suggested that two U:A:U base triples are formed in the RNA aptamer upon binding of ligands. Specific hydrogen bonds between arginine side chains of ligands and guanine bases located adjacent to the base triples are identified. On the basis of many intramolecular and intermolecular NOEs, a structural model of the complex has been constructed.     

Concerted motions in HIV-1 TAR RNA may allow access to bound state conformations: RNA dynamics from NMR residual dipolar couplings.

Ground-state dynamics in RNA is a critical precursor for structural adaptation observed ubiquitously in protein-RNA recognition. A tertiary conformational analysis of the stem-loop structural element in the transactivation response element (TAR) from human immunodeficiency virus type 1 (HIV-I) RNA is presented using recently introduced NMR methods that rely on the measurement of residual dipolar couplings (RDC) in partially oriented systems. Order matrix analysis of RDC data provides evidence for inter-helical motions that are of amplitude 46(+/-4) degrees, of random directional character, and that are executed about an average conformation with an inter-helical angle between 44 degrees and 54 degrees. The generated ensemble of TAR conformations have different organizations of functional groups responsible for interaction with the trans-activator protein Tat, including conformations similar to the previously characterized bound-state conformation. These results demonstrate the utility of RDC-NMR for simultaneously characterizing RNA tertiary dynamics and average conformation, and indicate an avenue for TAR complex formation involving tertiary structure capture.     

Inhibition of HIV-1 Tat-TAR interaction by diphenylfuran derivatives: effects of the terminal basic side chains.

A series of four biscationic diphenylfuran derivatives was used to investigate drug binding to the transactivation response element (TAR) RNA. The drugs, which are active against the Pneumocystis carinii pathogen (PCP), differ by the nature of the terminal basic side chains. Furimidazoline (DB60) is more potent at inhibiting binding of the Tat protein to TAR than furamidine (DB75) and the amidine-substituted analogues DB244 and DB226. In vivo studies using the fusion-induced gene stimulation (FIGS) assay entirely agree with the in vitro gel mobility shift data. The capacity of the drugs to antagonize Tat binding correlates with their RNA binding properties determined by melting temperature and RNase protection experiments. Footprinting studies indicate that the bulge region of TAR provides the identity element for the diphenylfurans. Access of the drugs to the major groove cavity at the pyrimidine bulge depends on the bulk of the alkylamine substituents. Experiments using TAR mutants show that the bulge of TAR is critical for drug binding but also reveal that the fit of the drugs into the major groove cavity of TAR does not involve specific contacts with the highly conserved residue U23 or the C x G26-39 base pair. The binding essentially involves shape recognition. The results are also discussed with respect to the known activity of the drug against PCP which is the major cause of mortality in AIDS patients. This study provides guidelines for future development of TAR-targeted anti-HIV-1 drugs.     

Dynamic ensemble view of the conformational landscape of HIV-1 TAR RNA and allosteric recognition

RNA conformational dynamics and the resulting structural heterogeneity play an important role in RNA functions, e.g., recognition. Recognition of HIV-1 TAR RNA has been proposed to occur via a conformational capture mechanism. Here, using ultrafast time-resolved fluorescence spectroscopy, we have probed the complexity of the conformational landscape of HIV-1 TAR RNA and monitored the position-dependent changes in the landscape upon binding of a Tat protein-derived peptide and neomycin B. In the ligand-free state, the TAR RNA samples multiple families of conformations with various degrees of base stacking around the three-nucleotide bulge region. Some subpopulations partially resemble those ligand-bound states, but the coaxially stacked state is below the detection limit. When Tat or neomycin B binds, the bulge region as an ensemble undergoes a conformational transition in a position-dependent manner. Tat and neomycin B induce mutually exclusive changes in the TAR RNA underlying the mechanism of allosteric inhibition at an ensemble level with residue-specific details. Time-resolved anisotropy decay measurements revealed picosecond motions of bases in both ligand-free and ligand-bound states. Mutation of a base pair at the bulge--stem junction has differential effects on the conformational distributions of the bulge bases. A dynamic model of the ensemble view of the conformational landscape for HIV-1 TAR RNA is proposed, and the implication of the general mechanism of RNA recognition and its impact on RNA-based therapeutics are discussed.     

Constructing RNA dynamical ensembles by combining MD and motionally decoupled NMR RDCs: new insights into RNA dynamics and adaptive ligand recognition

We describe a strategy for constructing atomic resolution dynamical ensembles of RNA molecules, spanning up to millisecond timescales, that combines molecular dynamics (MD) simulations with NMR residual dipolar couplings (RDC) measured in elongated RNA. The ensembles are generated via a Monte Carlo procedure by selecting snap-shot from an MD trajectory that reproduce experimentally measured RDCs. Using this approach, we construct ensembles for two variants of the transactivation response element (TAR) containing three (HIV-1) and two (HIV-2) nucleotide bulges. The HIV-1 TAR ensemble reveals significant mobility in bulge residues C24 and U25 and to a lesser extent U23 and neighboring helical residue A22 that give rise to large amplitude spatially correlated twisting and bending helical motions. Omission of bulge residue C24 in HIV-2 TAR leads to a significant reduction in both the local mobility in and around the bulge and amplitude of inter-helical bending motions. In contrast, twisting motions of the helices remain comparable in amplitude to HIV-1 TAR and spatial correlations between them increase significantly. Comparison of the HIV-1 TAR dynamical ensemble and ligand bound TAR conformations reveals that several features of the binding pocket and global conformation are dynamically preformed, providing support for adaptive recognition via a ‘conformational selection’ type mechanism.     

Ligand-induced changes in 2-aminopurine fluorescence as a probe for small molecule binding to HIV-1 TAR RNA

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated in part through an interaction between the virally encoded trans-activator protein Tat and the trans-activator responsive region (TAR) of the viral RNA genome. Because TAR is highly conserved and its interaction with Tat is required for efficient viral replication, it has received much attention as an antiviral drug target. Here, we report a 2-aminopurine (2-AP) fluorescence-based assay for evaluating potential TAR inhibitors. Through selective incorporation of 2-AP within the bulge (C23 or U24) of a truncated form of the TAR sequence (delta TAR-ap23 and delta TAR-ap24), binding of argininamide, a 24-residue arginine-rich peptide derived from Tat, and Neomycin has been characterized using steady-state fluorescence. Binding of argininamide to the 2-AP deltaTAR constructs results in a four- to 11-fold increase in fluorescence intensity, thus providing a sensitive reporter of that interaction (KD approximately 1 mM). Similarly, binding of the Tat peptide results in an initial 14-fold increase in fluorescence (KD approximately 25 nM), but is then followed by a slight decrease that is attributed to an additional, lower-affinity association(s). Using the deltaTAR-ap23 and TAR-ap24 constructs, two classes of Neomycin binding sites are detected; the first molecule of antibiotic binds as a noncompetitive inhibitor of Tat/argininamide (KD approximately 200 nM), whereas the second, more weakly bound molecule(s) becomes associated in a presumably nonspecific manner (KD approximately 4 microM). Taken together, the results demonstrate that the 2-AP fluorescence-detected binding assays provide accurate and general methods for quantitatively assessing TAR interactions.     

Molecular dynamics and binding specificity analysis of the bovine immunodeficiency virus BIV Tat-TAR complex

We have performed molecular dynamics (MD) simulations, with particle-mesh Ewald, explicit waters, and counterions, and binding specificity analyses using combined molecular mechanics and continuum solvent (MM-PBSA) on the bovine immunodeficiency virus (BIV) Tat peptide-TAR RNA complex. The solution structure for the complex was solved independently by Patel and co-workers and Puglisi and co-workers. We investigated the differences in both structures and trajectories, particularly in the formation of the U-A-U base triple, the dynamic flexibility of the Tat peptide, and the interactions at the binding interface. We observed a decrease in RMSD in comparing the final average RNA structures and initial RNA structures of both trajectories, which suggests the convergence of the RNA structures to a MD equilibrated RNA structure. We also calculated the relative binding of different Tat peptide mutants to TAR RNA and found qualitative agreement with experimental studies.