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.     

The bulge region of HIV-1 TAR RNA binds metal ions in solution

Binding of Mg²⁺, Ca²⁺ and Co(NH₃)₆³⁺ ions to the HIV-1 TAR RNA in solution was analysed by ¹⁹F NMR spectroscopy, metal ion-induced RNA cleavages and Brownian dynamics (BD) simulations. Chemically synthesised 29mer oligoribonucleotides of the TAR sequence labelled with 5-fluorouridine (FU) were used for ¹⁹F NMR-monitored metal ion titration. The chemical shift changes of fluorine resonances FU-23, FU-25 and FU-40 upon titration with Mg²⁺ and Ca²⁺ ions indicated specific, although weak, binding at the bulge region with the dissociation constants (K_d) of 0.9 ± 0.6 and 2.7 ± 1.7 mM, respectively. Argininamide, inducing largest ¹⁹F chemical shifts changes at FU-23, was used as a reference ligand (K_d = 0.3 ± 0.1 mM). In the Pb²⁺-induced TAR RNA cleavage experiment, strong and selective cleavage of the C24-U25 phosphodiester bond was observed, while Mg²⁺ and Ca²⁺ induced cuts at all 3-nt residues of the bulge. The inhibition of Pb²⁺-specific TAR cleavage by di- and trivalent metal ions revealed a binding specificity [in the order Co(NH₃)₆³⁺ > Mg²⁺ > Ca²⁺] at the bulge site. A BD simulation search of potential magnesium ion sites within the NMR structure of HIV-1 TAR RNA was conducted on a set of 20 conformers (PDB code 1ANR). For most cases, the bulge region was targeted by magnesium cations.     

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.     

反式激活应答元件RNA在HIV-1感染中的作用

反式激活应答(transactivation response,TAR)元件RNA作为HIV-1中的一种非编码RNA,从转录与翻译水平负调控HIV-1的基因表达.同时HIV-1采取了相应的策略拮抗TAR RNA的负调控作用:病毒蛋白Tat或细胞蛋白TAR RNA结合蛋白(TRBP)结合TAR RNA后,分别在转录与翻译水平促进HIV-1的基因表达.此外,TAR编码的miRNA有助于保持HIV的潜伏感染及阻止细胞凋亡.TAR与其它蛋白间相互作用及其功能的研究对于深入了解HIV-1感染细胞后的调控机制,寻求新的抗HIV治疗靶点具有重要意义.     

Solid-phase synthesis and thermal denaturation study of cyclic PNAs targeting the HIV-1 TAR RNA loop

Cyclic PNAs targeting the HIV-1 TAR RNA loop have been synthesized following a convenient solid-phase strategy which allows on-resin cyclisation. UV-monitored thermal denaturation studies demonstrate that these cyclic PNAs are able to strongly interact with their TAR RNA target, very likely through the formation of a six-base pair stable complex, involving the TAR RNA loop.     

Synthesis of site-specifically modified oligoribonucleotides for studies of the recognition of TAR RNA by HIV-1 tat protein and studies of hammerhead ribozymes.

Synthetic oligoribonucleotides having single uracil residues replaced by dU, dT, 2'-O-methylU or 5-bromodU have been prepared and used in the study of the interaction of HIV-1 tat protein with an RNA stem-loop. The preparation of phosphoramidites of 5-bromouridine and purine riboside suitable for use in solid-phase oligoribonucleotide synthesis is also described. The effect of adenine replacement by purine in a hammerhead ribozyme has also been determined.     

Detailed mutational analysis of TAR RNA: critical spacing between the bulge and loop recognition domains.

Trans-activation of HIV-1 by the Tat protein is mediated through a cis-acting element (TAR) in the viral RNA. In order to obtain further insight into the molecular interactions for trans-activation, a detailed mutational analysis of TAR RNA was carried out. TAR RNA forms a hairpin structure with important sequence elements in the single-stranded bulge- and loop-domains. We found that the sequence of the base-pairs flanking the bulge is critical for Tat-mediated trans-activation. In addition, Tat-response is reduced when the bulge is forced into a base-paired configuration through the introduction of complementary nucleotides on the opposite side of the stem. Thus, the 3-nucleotide bulge and adjacent base-pairs comprise a recognition domain with both sequence- and structure-elements. Accessibility of the loop sequences is also important for Tat function, since base-pairing through the formation of a pseudoknot-like structure does inhibit Tat action. A third critical parameter that influences the magnitude of Tat response is the number of loop nucleotides. Finally, the relative spacing between the loop and the bulge is also important. We introduced additional base-pairs in the stem connecting the two domains. Such mutations progressively decreased the efficiency of Tat induction. Interestingly, activity of the HIV-2 Tat protein did markedly increase on targets with one or two additional basepairs. These results suggest that Tat interacts with a cellular loop-binding protein(s) to increase HIV gene expression.     

RNA bulge entropies in the unbound state correlate with peptide binding strengths for HIV-1 and BIV TAR RNA because of improved conformational access.

For the binding of peptides to wild-type HIV-1 and BIV TAR RNA and to mutants with bulges of various sizes, changes in the DeltaDelta G values of binding were determined from experimental K d values. The corresponding entropies of these bulges are estimated by enumerating all possible RNA bulge conformations on a lattice and then applying the Boltzmann relationship. Independent calculations of entropies from fluctuations are also carried out using the Gaussian network model (GNM) recently introduced for analyzing folded structures. Strong correlations are seen between the changes in free energy determined for binding and the two different unbound entropy calculations. The fact that the calculated entropy increase with larger bulge size is correlated with the enhanced experimental binding free energy is unusual. This system exhibits a dependence on the entropy of the unbound form that is opposite to usual binding models. Instead of a large initial entropy being unfavorable since it would be reduced upon binding, here the larger entropies actually favor binding. Several interpretations are possible: (i) the higher conformational freedom implies a higher competence for binding with a minimal strain, by suitable selection amongst the set of already accessible conformations; (ii) larger bulge entropies enhance the probability of the specific favorable conformation of the bound state; (iii) the increased freedom of the larger bulges contri-butes more to the bound state than to the unbound state; (iv) indirectly the large entropy of the bound state might have an unfavorable effect on the solvent structure. Nonetheless, this unusual effect is interesting.     

TAR RNA结合蛋白在HIV-1感染中的作用

TARRNA结合蛋白是细胞中双链RNA结合蛋白家族成员之一.它可以结合HIV-1TARRNA,并与Tat协同作用激活LTR表达,进而促进病毒的转录与翻译.TRBP也是将干扰素抗病毒通路与RNA干扰免疫通路相连的一种细胞蛋白.在干扰素诱生的PKR反应中,TRBP通过直接抑制PKR的自磷酸化、与PKR竞争通用的RNA底物或与PACT形成异源二聚体等机制抑制细胞内的PKR反应,从而降低了PKR介导的对病毒表达的抑制作用.TRBP与Dicer和Ago2等组成的RNA诱导沉默复合体,在RNA干扰中发挥着关键作用并调控随后的序列特异性降解.在HIV-1感染中,TRBP更倾向于促进病毒的表达与复制,因此TRBP也成为控制HIV-1感染的新靶点.     

Thermodynamic examination of trinucleotide bulged RNA in the context of HIV-1 TAR RNA

RNA structures contain many bulges and loops that are expected to be sites for inter- and intra-molecular interactions. Nucleotides in the bulge are expected to influence the structure and recognition of RNA. The same stability is assigned to all trinucleotide bulged RNA in the current secondary structure prediction models. In this study thermal denaturation experiments were performed on four trinucleotide bulged RNA, in the context of HIV-1 TAR RNA, to determine whether the bulge sequence affects RNA stability and its divalent ion interactions. Cytosine-rich bulged RNA were more stable than uracil-rich bulged RNA in 1 M KCl. Interactions of divalent ions were more favorable with uracil-rich bulged RNA by ~2 kcal/mol over cytosine-rich bulged RNA. The UCU-TAR RNA (wild type) is stabilized by 1.7 kcal/mol in 9.5 mM Ca²⁺ as compared with 1 M KCl, whereas no additional gain in stability is measured for CCC-TAR RNA. These results have implications for base substitution experiments traditionally employed to identify metal ion binding sites. To our knowledge, this is the first systematic study to quantify the effect of small sequence changes on RNA stability upon interactions with divalent ions.     

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.