Identification of a novel HIV-1 TAR RNA bulge binding protein.

The Tat protein binds to TAR RNA to stimulate the expression of the human immunodeficiency virus type 1 (HIV-1) genome. Tat is an 86 amino acid protein that contains a short region of basic residues (aa49-aa57) that are required for RNA binding and TAR is a 59 nucleotide stem-loop with a tripyrimidine bulge in the upper stem. TAR is located at the 5' end of all viral RNAs. In vitro, Tat specifically interacts with TAR by recognising the sequence of the bulge and upper stem, with no requirement for the loop. However, in vivo the loop sequence is critical for activation, implying a requirement for accessory cellular TAR RNA binding factors. A number of TAR binding cellular factors have been identified in cell extracts and various models for the function of these factors have been suggested, including roles as coactivators and inhibitors. We have now identified a novel 38 kD cellular factor that has little general, single-stranded or double-stranded RNA binding activity, but that specifically recognises the bulge and upper stem region of TAR. The protein, referred to as BBP (bulge binding protein), is conserved in mammalian and amphibian cells and in Schizosaccharomyces pombe but is not found in Saccharomyces cerevisiae. BBP is an effective competitive inhibitor of Tat binding to TAR in vitro. Our data suggest that the bulge-stem recognition motif in TAR is used to mediate cellular factor/RNA interactions and indicates that Tat action might be inhibited by such competing reactions in vivo.     

Effect of different arginine methylations on the thermodynamics of Tat peptide binding to HIV-1 TAR RNA

RNA-binding proteins are an important class of mediators that regulate cell function and differentiation. Methylation of arginine, a post-translational modification (PTM) found in these proteins, can modulate their function. Arginine can be monomethylated or dimethylated, depending on the type of methyl transferases involved. This paper describes a comparative study of the thermodynamics of unmodified and modified Tat peptide interaction with TAR RNA, where the peptide is methylated at epsilon (?) and eta (η) nitrogen atoms of guanidinium group of arginine side chain at position 52 or 53. The results indicate that monomethylation of arginine at epsilon (?) nitrogen atom enhances binding affinity, owing to a more favourable enthalpy component which overrides the less favourable entropy change. In contrast, monomethylation of arginine residue at η nitrogen results in reduced binding affinity originating exclusively from a less favourable enthalpy change leaving entropic component unaffected. However, in case of simultaneous methylation at ? and η positions, the binding parameters remain almost unaffected, when compared to the unmodified peptide. In case of symmetric dimethylation at η position the observed enthalpy change of the binding was found to be smaller than the values obtained for the unmodified peptide. Asymmetric dimethylation at η position showed the most reduced binding affinities owing to less favourable enthalpy changes. These results provide insights that enable elucidation of the biological outcome of arginine methylation as PTMs that regulate protein function, and will contribute to our understanding of how these PTMs are established in vitro and in vivo.     

Polyamidoamine dendrimers inhibit binding of Tat peptide to TAR RNA

The binding of polyamidoamine (PAMAM) dendrimer or Tat peptide to trans-acting responsive element (TAR) RNA has been studied using microgravimetric quartz crystal microbalance (QCM). Experimental results showed that PAMAM dendrimer could form complexes with TAR RNA. Especially, PAMAM dendrimer could disrupt the interaction of Tat peptide with TAR RNA, which is essential for HIV-1 virus replication, suggesting that QCM is a powerful tool for studying the binding processes of Tat peptide-TAR RNA and drug-TAR RNA and has great significance for the design of new drugs. An equation to measure the binding ability between TAR RNA and other species has been proposed.     

Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat.

Human cyclin T1 (hCycT1), a major subunit of the essential elongation factor P-TEFb, has been proposed to act as a cofactor for human immunodeficiency virus type 1 (HIV-1) Tat. Here, we show that murine cyclin T1 (mCycT1) binds the activation domain of HIV-1 Tat but, unlike hCycT1, cannot mediate Tat function because it cannot be recruited efficiently to TAR. In fact, overexpression of mCycT1, but not hCycT1, specifically inhibits Tat-TAR function in human cells. This discordant phenotype results from a single amino acid difference between hCycT1 and mCycT1, a tyrosine in place of a cysteine at residue 261. These data indicate that the ability of Tat to recruit CycT1/P-TEFb to TAR determines the species restriction of HIV-1 Tat function in murine cells and therefore demonstrate that this recruitment is a critical function of the Tat protein.     

Characterization of the solution conformations of unbound and Tat peptide-bound forms of HIV-1 TAR RNA.

Basic peptides from the carboxy terminus of the HIV-1 Tat protein bind to the apical stem-loop region of TAR RNA with high affinity and moderate specificity. The conformations of the unbound and 24 residue Tat peptide (Tfr24)-bound forms of TAR RNA have been characterized by NMR spectroscopy. The unbound form of TAR exists in major and minor forms having different trinucleotide bulge conformations. A specific TAR RNA conformational change is observed upon complex formation with Tfr24, consisting of coaxial stacking of helical stems and base triple formation. A U23-A27-U38 base triple is proposed based on exchangeable proton NMR data, where U23 forms a base pair with A27 in the major groove. No evidence for base triple formation was found for Tat peptides in which lysine residues are extensively substituted for arginine.     

HIV-1 TAR RNA subverts RNA interference in transfected cells through sequestration of TAR RNA-binding protein, TRBP

TAR RNA-binding protein, TRBP, was recently discovered to be an essential partner for Dicer and a crucial component of the RNA-induced silencing complex (RISC), a critical element of the RNA interference (RNAi) of the cell apparatus. Human TRBP was originally characterized and cloned 15 years ago based on its high affinity for binding the HIV-1 encoded leader RNA, TAR. RNAi is used, in part, by cells to defend against infection by viruses. Here, we report that transfected TAR RNA can attenuate the RNAi machinery in human cells. Our data suggest that TAR RNA sequesters TRBP rendering it unavailable for downstream Dicer-RISC complexes. TAR-induced inhibition of Dicer-RISC activity in transfected cells was partially relieved by exogenous expression of TRBP.     

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.     

Oligonucleotide inhibition of the interaction of HIV-1 Tat protein with the trans-activation responsive region (TAR) of HIV RNA

The interaction of HIV-1 Tat protein with its recognition sequence, the trans-activation responsive region TAR is a potential target for drug discovery against HIV infection. We show by use of an in vitro competition filter binding interference assay that synthetic oligodeoxyribonucleotides complementary to the HIV-1 TAR RNA apical stem-loop and bulge region inhibit the binding of Tat protein or a Tat peptide (residues 37-72) better than two small molecules that have been shown to bind TAR RNA, Hoechst 33258 and neomycin B. The inhibition is not sensitive to length between 13 and 16 residues or precise positioning but shorter oligonucleotides are less effective. Enhanced inhibition was obtained for a 16-mer 2'-O-methyl oligoribonucleotide but not for C5-propyne pyrimidine-substituted oligonucleotides. Control non-antisense oligonucleotides were occasionally also effective in filter binding interference but only the complementary antisense 2'-O-methyl oligoribonucleotide was effective in gel mobility shift assays in direct TAR binding or in interference with Tat peptide binding to the TAR stem-loop. This is the first demonstration of effective inhibition of the Tat-TAR interaction by nuclease-stabilized oligonucleotide analogues.     

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.     

Using pyrene-labeled HIV-1 TAR to measure RNA-small molecule binding

To quantitatively understand the binding affinity and target selectivity of small-molecule RNA interactions, it is useful to have a rapid, highly reproducible binding assay that can be readily generalized to different RNA targets. To that end, an assay has been developed and validated for measuring the binding of low-molecular weight ligands to RNA by monitoring the fluorescence of a covalently incorporated fluorophore. As a test system, the fluorescence of a pyrene-derivatized HIV-1 TAR (transactivating response element) RNA was measured upon titration with aminoglycoside antibiotics. The binding isotherms thus obtained fit well with a model for a 1:1 interaction and yield an accurate measure of the equilibrium dissociation constant. Among a series of natural aminoglycosides, the binding affinity correlates with the number of amines, supporting an electrostatic compensation model for binding. Furthermore, the ionic strength dependence confirms that much of the binding energy is electrostatic. Finally, by measuring the binding affinity in the presence of nucleic acid competitors, we confirm that although aminoglycosides show high RNA to DNA selectivity, their selectivity among different RNA targets is sub- optimal. We conclude that this newly developed assay can be generalized to measure the binding affinities and selectivities of a variety of small molecules to a specific RNA target.     

TAR-RNA binding by HIV-1 Tat protein is selectively inhibited by its L-enantiomer.

An oligoribonucleotide, corresponding to the Tat-interactive top half of the HIV-1 TAR RNA stem-loop, was synthesized in both the natural D- and the enantiomeric L-configurations. The affinity of Tat for the two RNAs, assessed by competition binding experiments, was found to be identical and is reduced 10-fold for both, upon replacement of the critical bulge residue U23 with cytidine. It is suggested that this interaction of the flexible Tat protein depends strongly upon the tertiary structure of a binding pocket within TAR, but not upon its handedness, and may be described by a 'hand-in-mitten' model.