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.     

Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein

The pharmacological disruption of the interaction between the HIV Tat protein and its cognate transactivation response RNA (TAR) would generate novel anti-viral drugs with a low susceptibility to drug resistance, but efforts to discover ligands with sufficient potency to warrant pharmaceutical development have been unsuccessful. We have previously described a family of structurally constrained β-hairpin peptides that potently inhibits viral growth in HIV-infected cells. The nuclear magnetic resonance (NMR) structure of an inhibitory complex revealed that the peptide makes intimate contacts with the 3-nt bulge and the upper helix of the RNA hairpin, but that a single residue contacts the apical loop where recruitment of the essential cellular co-factor cyclin T₁ occurs. Attempting to extend the peptide to form more interactions with the RNA loop, we examined a library of longer peptides and achieved >6-fold improvement in affinity. The structure of TAR bound to one of the extended peptides reveals that the peptide slides down the major groove of the RNA, relative to our design, in order to maintain critical interactions with TAR. These conserved contacts involve three amino acid side chains and identify critical interaction points required for potent and specific binding to TAR RNA. They constitute a template of essential interactions required for inhibition of this RNA.     

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.