Refolded HIV-1 tat protein protects both bulge and loop nucleotides in TAR RNA from ribonucleolytic cleavage

Substantial evidence indicates that HIV-1 trans-activation by tat protein is mediated through the TAR RNA element. This RNA forms a stem-loop structure containing a three-nucleotide bulge and a six-nucleotide loop. Previous mutagenic analysis of TAR indicates that the bulge residues and a 4 bp segment of the stem constitute, in part, the tat binding site. However, there appears to be no sequence-specific contribution of the six-base loop. We have employed a ribonuclease protection technique to explore the interaction of tat with single-stranded regions of TAR. The results indicate that tat interacts with both the bulge and loop regions of TAR. Treatment of TAR RNA with RNase A results in cleavage at U23 and U31, located in the bulge and loop regions, respectively. High concentrations (approximately 2 microM) of Escherichia coli derived tat protein, prepared by standard procedures, gave complete protection of TAR RNA from RNase A cleavage. However, under these conditions, truncated TAR derivatives in which no stem-loop structure is expected to form were also protected, indicating nonspecific binding. In order to obtain a tat preparation with enhanced specificity toward TAR RNA, methods were developed for refolding the recombinant protein. This treatment enhanced the affinity of tat for TAR by approximately 30-fold [Kd(apparent) less than 25 nM] and markedly increased its specificity for the TAR. Again, tat protected TAR RNA from RNase A cleavage at both U23 and U31. Protection was also observed with RNase T1 which cleaves TAR RNA at three G residues in the six-base loop.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

High affinity RNA for mammalian initiation factor 4E interferes with mRNA-cap binding and inhibits translation

The eukaryotic translation initiation factor 4F (eIF4F) consists of three polypeptides (eIF4A, eIF4G, and eIF4E) and is responsible for recruiting ribosomes to mRNA. eIF4E recognizes the mRNA 5'-cap structure (m7GpppN) and plays a pivotal role in control of translation initiation, which is the rate-limiting step in translation. Overexpression of eIF4E has a dramatic effect on cell growth and leads to oncogenic transformation. Therefore, an inhibitory agent to eIF4E, if any, might serve as a novel therapeutic against malignancies that are caused by aberrant translational control. Along these lines, we developed two RNA aptamers, aptamer 1 and aptamer 2, with high affinity for mammalian eIF4E by in vitro RNA selection-amplification. Aptamer 1 inhibits the cap binding to eIF4E more efficiently than the cap analog m7GpppN or aptamer 2. Consistently, aptamer 1 inhibits specifically cap-dependent in vitro translation while it does not inhibit cap-independent HCV IRES-directed translation initiation. The interaction between eIF4E and eIF4E-binding protein 1 (4E-BP1), however, was not inhibited by aptamer 1. Aptamer 1 is composed of 86 nucleotides, and the high affinity to eIF4E is affected by deletions at both termini. Moreover, relatively large areas in the aptamer 1 fold are protected by eIF4E as determined by ribonuclease footprinting. These findings indicate that aptamers can achieve high affinity to a specific target protein via global conformational recognition. The genetic mutation and affinity study of variant eIF4E proteins suggests that aptamer 1 binds to eIF4E adjacent to the entrance of the cap-binding slot and blocks the cap-binding pocket, thereby inhibiting translation initiation.     

Cation-dependent folding of 3′ cap-independent translation elements facilitates interaction of a 17-nucleotide conserved sequence with eIF4G

The 3′-untranslated regions of many plant viral RNAs contain cap-independent translation elements (CITEs) that drive translation initiation at the 5′-end of the mRNA. The barley yellow dwarf virus-like CITE (BTE) stimulates translation by binding the eIF4G subunit of translation initiation factor eIF4F with high affinity. To understand this interaction, we characterized the dynamic structural properties of the BTE, mapped the eIF4G-binding sites on the BTE and identified a region of eIF4G that is crucial for BTE binding. BTE folding involves cooperative uptake of magnesium ions and is driven primarily by charge neutralization. Footprinting experiments revealed that functional eIF4G fragments protect the highly conserved stem–loop I and a downstream bulge. The BTE forms a functional structure in the absence of protein, and the loop that base pairs the 5′-untranslated region (5′-UTR) remains solvent-accessible at high eIF4G concentrations. The region in eIF4G between the eIF4E-binding site and the MIF4G region is required for BTE binding and translation. The data support the model in which the eIF4F complex binds directly to the BTE which base pairs simultaneously to the 5′-UTR, allowing eIF4F to recruit the 40S ribosomal subunit to the 5′-end.     

A novel inhibitor of cap-dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E.

In the yeast Saccharomyces cerevisiae a small protein named p20 is found associated with translation initiation factor eIF4E, the mRNA cap-binding protein. We demonstrate here that p20 is a repressor of cap-dependent translation initiation. p20 shows amino acid sequence homology to a region of eIF4G, the large subunit of the cap-binding protein complex eIF4F, which carries the binding site for eIF4E. Both, eIF4G and p20 bind to eIF4E and compete with each other for binding to eIF4E. The eIF4E-p20 complex can bind to the cap structure and inhibit cap-dependent but not cap-independent translation initiation: the translation of a mRNA with the 67 nucleotide omega sequence of tobacco mosaic virus in its 5' untranslated region (which was previously shown to render translation cap-independent) is not inhibited by p20. Whereas the translation of the same mRNA lacking the omega sequence is strongly inhibited by p20. Disruption of CAF20, the gene encoding p20, stimulates the growth of yeast cells, overexpression of p20 causes slower growth of yeast cells. These results show that p20 is a regulator of eIF4E activity which represses cap-dependent initiation of translation by interfering with the interaction of eIF4E with eIF4G, e.g. the formation of the eIF4F-complex.     

The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA

RNA dimerization is an essential step in the retroviral life cycle. Dimerization and encapsidation signals, closely linked in HIV-2, are located in the leader RNA region. The SL1 motif and nucleocapsid protein are considered important for both processes. In this study, we show the structure of the HIV-2 leader RNA (+1-560) captured as a loose dimer. Potential structural rearrangements within the leader RNA were studied. In the loose dimer form, the HIV-2 leader RNA strand exists in vitro as a single global fold. Two kissing loop interfaces within the loose dimer were identified: SL1/SL1 and TAR/TAR. Evidence for these findings is provided by RNA probing using SHAPE, chemical reagents, enzymes, non-denaturing PAGE mobility assays, antisense oligonucleotides hybridization and analysis of an RNA mutant. Both TAR and SL1 as isolated domains are bound by recombinant NCp8 protein with high affinity, contrary to the hairpins downstream of SL1. Foot-printing of the SL1/NCp8 complex indicates that the major binding site maps to the SL1 upper stem. Taken together, these data suggest a model in which TAR hairpin III, the segment of SL1 proximal to the loop and the PAL palindromic sequence play specific roles in the initiation of dimerization.     

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 presence of the TAR RNA structure alters the programmed -1 ribosomal frameshift efficiency of the human immunodeficiency virus type 1 (HIV-1) by modifying the rate of translation initiation

HIV-1 uses a programmed -1 ribosomal frameshift to synthesize the precursor of its enzymes, Gag-Pol. The frameshift efficiency that is critical for the virus replication, is controlled by an interaction between the ribosome and a specific structure on the viral mRNA, the frameshift stimulatory signal. The rate of cap-dependent translation initiation is known to be altered by the TAR RNA structure, present at the 5′ and 3′ end of all HIV-1 mRNAs. Depending upon its concentration, TAR activates or inhibits the double-stranded RNA-dependent protein kinase (PKR). We investigated here whether changes in translation initiation caused by TAR affect HIV-1 frameshift efficiency. CD4+ T cells and 293T cells were transfected with a dual-luciferase construct where the firefly luciferase expression depends upon the HIV-1 frameshift. Translation initiation was altered by adding TAR in cis or trans of the reporter mRNA. We show that HIV-1 frameshift efficiency correlates negatively with changes in the rate of translation initiation caused by TAR and mediated by PKR. A model is presented where changes in the rate of initiation affect the probability of frameshifting by altering the distance between elongating ribosomes on the mRNA, which influences the frequency of encounter between these ribosomes and the frameshift stimulatory signal.     

trans activation of human immunodeficiency virus type 1 is sequence specific for both the single-stranded bulge and loop of the trans-acting-responsive hairpin: a quantitative analysis.

We have used site-directed mutagenesis to delineate sequence specific domains within the human immunodeficiency virus type 1 (HIV-1) trans-acting-responsive (TAR) RNA element that are required for trans activation by the viral Tat protein. Our data in part corroborate a recent report [S. Feng and E. C. Holland, Nature (London) 334:165-167, 1988] that five nucleotides within the loop (+29 to +33) of the TAR hairpin are important for trans activation. We, however, found no absolute requirement for the CUGGG loop sequence. Mutants with substitutions within the loop retained between 9 and 50% activity compared with the wild type. A second sequence, important for trans activation, was found in the 3-base bulge loop (+22 to +24) of the TAR hairpin. Cross-trans-activation studies of mutant HIV-1 TAR elements with the HIV-2 Tat protein suggest that a similar recognition event(s) forms the basis for trans activation of HIV-1 and HIV-2.     

Activation of double-stranded RNA-dependent kinase (dsl) by the TAR region of HIV-1 mRNA: a novel translational control mechanism

All mRNAs of human immunodeficiency virus 1 (HIV-1) contain in their 5' untranslated region a sequence termed TAR that responds to trans-activation by the tat (trans-activating) protein. This RNA sequence assumes a stable secondary structure, and its cap structure is relatively inaccessible. Here we report that these structural properties of the TAR sequence underlie the ability of TAR to inhibit in trans the translation of other mRNAs. This mechanism of translation inhibition involves the activation of the double-stranded RNA-dependent kinase (dsl), which in turn phosphorylates the protein synthesis initiation factor 2 (eIF-2). Mutations in the TAR region that diminish the stability of the secondary structure cause a significant reduction in the trans-inhibition. A similar reduction in the dsl activation occurs when TAR is placed further downstream of the cap structure. This is a clear demonstration of a specific naturally occurring mRNA sequence that can activate dsl. We suggest a novel translational regulatory mechanism that interdigitates the activities of eIF-2 and eIF-4F.     

A small stem loop element directs internal initiation of the URE2 internal ribosome entry site in Saccharomyces cerevisiae

Internal initiation of translation is the process of beginning protein synthesis independent of the m(7)G cap structure at the 5'-end of an mRNA molecule. We have previously shown that the URE2 mRNA in the yeast Saccharomyces cerevisiae contains an internal ribosome entry site (IRES) whose activity is suppressed by eukaryotic initiation factor 2A (eIF2A; YGR054W). In this study, the minimal sequence required to efficiently direct internal initiation was determined using a system that abrogates cap-dependent scanning of the 40 S ribosomal subunit in both wild-type and eIF2A knock-out cells. Subsequently, secondary structural elements within the minimal sequence were determined by probing with RNases T1 and V1 and the small molecule diethylpyrocarbonate. It was found that the URE2 minimal IRES comprises a 104 nucleotide A-rich stem loop element encompassing the internal AUG codon. Interestingly, the internal AUG seems to be involved in base-pairing interactions that would theoretically hamper its ability to interact with incoming initiator tRNA molecules. Furthermore, none of the truncations used to identify the minimal IRES element were capable of abrogating the suppressive effect of eIF2A. Our data provide the first insight into the RNA structural requirements of the yeast translational machinery for cap-independent initiation of protein synthesis.     

Orientation-specific cis complementation by bulge- and loop-mutated human immunodeficiency virus type 1 TAR RNAs.

Tat activates human immunodeficiency type 1 gene expression by binding to TAR RNA. TAR comprises a partially base paired stem and hexanucleotide loop with a tripyrimidine bulge in the upper stem. In vitro, Tat binds to the bulge and upper stem, with no requirement for the loop. However, in vivo, loop sequences are critical for activation, implying that a loop binding cellular factor may be involved in the activation pathway. Given that activation appears to be a two-component system comprising a Tat-bulge interaction and a cellular factor-loop interaction, we considered that it might be possible to spatially separate the two components and retain activation. We have constructed a series of double TAR elements comprising various combinations of mutated TAR structures. Defective TARs with nucleotide substitutions in either the bulge or the loop complemented each other to give wild-type activation. However, the complementation was orientation specific, requiring the intact Tat binding site to reside on the 5'-proximal TAR. These data suggest that provided the wild-type orientation of the bulge and loop elements is retained, there is no requirement for them to coexist on the same TAR structure.     

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.