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.     

Secondary structure of the human T-cell leukemia virus type 1 rex-responsive element is essential for rex regulation of RNA processing and transport of unspliced RNAs.

Rex protein of human T-cell leukemia virus type 1 (HTLV-1) induces cytoplasmic expression of unspliced gag/pol mRNA and singly spliced env mRNA and thus is essential for replication of the virus. This regulation requires a cis-acting rex-responsive element (RXE), located in the 3' region of the viral RNA. By external deletion, we have identified RXE composed of 205 nucleotides. The secondary structure of RXE was confirmed by studies on its susceptibility to nuclease digestions to consist of four stem-loops and a long stretch of stem structure. Substitution and deletion mutations revealed that two regions of the stem-loops and their secondary structures are essential for rex regulation. Similar secondary structures were found in the corresponding regions of HTLV-2, bovine leukemia virus and human immunodeficiency virus. Furthermore, a sequence of 11 nucleotides in the RXE was found to be conserved in the secondary structures of HTLV-1, HTLV-2, and bovine leukemia virus. These observations suggest that the secondary structure as well as the conserved sequence may be important in expression of unspliced RNA even with diverged sequences as observed in these viruses.     

Characterization of the nuclear export signal of human T-cell lymphotropic virus type 1 Rex reveals that nuclear export is mediated by position-variable hydrophobic interactions.

We previously determined that amino acids 64 to 120 of human T-cell lymphotropic virus type 1 (HTLV-1) Rex can restore the function of an effector domain mutant of human immunodeficiency virus type 1 (HIV-1) Rev (T. J. Hope, B. L. Bond, D. McDonald, N. P. Klein, and T. G. Parslow, J. Virol. 65:6001-6007, 1991). In this report, we (i) identify and characterize a position-independent 17-amino-acid region of HTLV-1 Rex that fully complements HIV-1 Rev effector domain mutants and (ii) show that this 17-amino-acid region and specific hydrophobic substitutions can serve as nuclear export signals. Mutagenesis studies revealed that four leucines within the minimal region were essential for function. Alignment of the minimal Rex region with the HIV-1 Rev effector domain suggested that the position of some of the conserved leucines is flexible. We found two of the leucines could each occupy one of two positions within the context of the full-length HTLV-1 Rex protein and maintain function. The idea of flexibility within the Rex effector domain was confirmed and extended by identifying functional substitutions by screening a library of effector domain mutants in which the two regions of flexibility were randomized. Secondly, the functional roles of the minimal Rex effector domain and hydrophobic substitutions were independently confirmed by demonstrating that these effector domains could serve as nuclear export signals when conjugated with bovine serum albumin. Nuclear export of the wild-type Rex conjugates was temperature dependent and sensitive to wheat germ agglutinin and was blocked by a 20-fold excess of unlabeled conjugates. Together, these studies reveal that position-variable hydrophobic interactions within the HTLV-1 Rex effector domain mediate nuclear export function.     

Specific labeling approaches to guanine and adenine imino and amino proton assignments in the AMP–RNA aptamer complex

The secondary structure of a recently identified ATP-binding RNA aptamer consists of apurine-rich 11-residue internal loop positioned opposite a single guanine bulge flanked oneither side by helical stem segments. The ATP ligand targets the internal loop and bulgedomains, inducing a structural transition in this RNA segment on complex formation.Specifically, 10 new slowly exchanging proton resonances in the imino, amino and sugarhydroxyl chemical shift range are observed on AMP–RNA aptamer complex formation.This paper outlines site-specific labeling approaches to identify slowly exchanging imino(guanine) and amino (guanine and adenine) protons in internal loop and bulge segments ofcompact RNA folds such as found in the AMP–RNA aptamer complex. One approachincorporates 15N-labeled guanine (N1 imino and N2 amino positions) and 15N-labeledadenine (N6 amino position), one residue at a time, in the AMP-binding RNA aptamer, withlabeling incorporation through chemical synthesis facilitated by generating the aptamer fromtwo separate strands. The unambiguous assignments deduced from the 15N labeling studieshave been verified from an independent labeling strategy where individual guanines in theinternal loop have been replaced, one at a time, by inosines and assignments were made onthe basis of the large 2 ppm downfield shift of the guanine imino protons on inosinesubstitution. The strengths and limitations of the inosine-for-guanine substitution approachemerge from our studies on the AMP–RNA aptamer complex. The assignment of theinternal loop and bulge imino and amino protons was critical in our efforts to define thesolution structure of the AMP–RNA aptamer complex since these slowly exchangingprotons exhibit a large number of long-range intramolecular NOEs within the RNA, as wellas intermolecular NOEs to the AMP in the complex. The current application of specific 15Nand inosine labeling approaches for exchangeable imino and amino proton assignments in thenonhelical segments of an RNA aptamer complex in our laboratory complements selective 2Hand 13C approaches to assign nonexchangeable base and sugar protons in RNA andligand–RNA complexes reported in the literature.     

Multimer Formation Is Not Essential for Nuclear Export of Human T-Cell Leukemia Virus Type 1 Rex trans-Activator Protein

The Rex trans-regulatory protein of human T-cell leukemia virus type 1 (HTLV-1) is required for the nuclear export of incompletely spliced and unspliced viral mRNAs and is therefore essential for virus replication. Rex is a nuclear phosphoprotein that directly binds to its cis-acting Rex response element RNA target sequence and constantly shuttles between the nucleus and cytoplasm. Moreover, Rex induces nuclear accumulation of unspliced viral RNA. Three protein domains which mediate nuclear import-RNA binding, nuclear export, and Rex oligomerization have been mapped within the 189-amino-acid Rex polypeptide. Here we identified a different region in the carboxy-terminal half of Rex which is also required for biological activity. In inactive mutants with mutations that map within this region, as well as in mutants that are deficient in Rex-specific multimerization, Rex trans activation could be reconstituted by fusion to a heterologous leucine zipper dimerization interface. The intracellular trafficking capabilities of wild-type and mutant Rex proteins reveal that biologically inactive and multimerization-deficient Rex mutants are still efficiently translocated from the nucleus to the cytoplasm. This observation indicates that multimerization is essential for Rex function but is not required for nuclear export. Finally, we are able to provide an improved model of the HTLV-1 Rex domain structure.     

Is unphosphorylated Rex,as multifunctional protein of HTLV-1, a fully intrinsically disordered protein? An in silico study

Intracellularlocation of a viral unspliced mRNA in host cell is a crucial factor for normal life of the virus. Rex is a neucleo-cytoplasmic shuffling protein of Human T-cell Leukemia Virus-1(HTLV-1)which has important role in active transport of cargo-containing RNA from nucleus to cytoplasm. Therefore, it plays a crucial role in the disease development by the virus. In spite of its importance, the 3d-structurephosphorylated and unphosphorylated of this protein has not been determined. In this study, first we predicted whether Rex protein is an ordered or disordered protein. In second step protein 3Dstructure of Rex was obtained. The content of disorder-promoting amino acids, flexibility, hydrophobicity, short linear motifs (SLiMs) and protein binding regions and probability of Rex crystallization were calculated by various In Silico methods. The3D models of Rex protein were obtained by various In Silico methods, such as homology modeling, threading and ab initio, including; I-TASSER, LOMETS, SPARSKS, ROBBETA and QUARK servers. By comparing and analyzing Qmean, z-scores and energy levels of selected models, the best structures with highest favored region in Ramachandran plot (higher than 90%) was refined with MODREFINER software. In silico analysis of Rex physicochemical properties and also predicted SLiMs and binding regions sites confirms that unphosphorylated Rex protein in HTLV-1 as Rev protin in HIV is wholly disordered protein belongs to the class of intrinsically disordered proteins with extended disorder (native coils, native pre-molten globules).     

Arginine-rich motifs present multiple interfaces for specific binding by RNA

A number of proteins containing arginine-rich motifs (ARMs) are known to bind RNA and are involved in regulating RNA processing in viruses and cells. Using automated selection methods we have generated a number of aptamers against ARM peptides from various natural proteins. Aptamers bind tightly to their cognate ARMs, with K(d) values in the nanomolar range, and frequently show no propensity to bind to other ARMs or even to single amino acid variants of the cognate ARM. However, at least some anti-ARM aptamers can cross-recognize a limited set of other ARMs, just as natural RNA-binding sites have been shown to exhibit so-called "chameleonism." We expand upon the number of examples of cross-recognition and, using mutational and circular dichroism (CD) analyses, demonstrate that there are multiple mechanisms by which RNA ligands can cross-recognize ARMs. These studies support a model in which individual arginine residues govern binding to an RNA ligand, and the inherent flexibility of the peptide backbone may make it possible for "semi-specific" recognition of a discrete set of RNAs by a discrete set of ARM peptides and proteins.     

Assignment and modeling of the Rev Response Element RNA bound to a Rev peptide using 13C-heteronuclear NMR

Summary The Rev Response Element (RRE) RNA-Rev protein interaction is important for regulation of gene expression in the human immunodeficiency virus. A model system for this interaction, which includes stem IIB of the RRE RNA and an arginine-rich peptide from the RNA-binding domain of Rev, was studied using multidimensional heteronuclear NMR. Assignment of the RNA when bound to the peptide was obtained from NMR experiments utilizing uniformly and specifically ¹³C-labeled RNA. Isotopic filtering experiments on the specifically labeled RNA enabled unambiguous assignment of unusual nonsequential NOE patterns present in the internal loop of the RRE. A three-dimensional model of the RNA in the complex was obtained using restrained molecular dynamics calculations. The internal loop contains two purine-purine base pairs, which are stacked to form one continuous helix flanked by two A-form regions. The formation of a G-G base pair in the internal loop requires an unusual structure of the phosphate backbone. This structural feature is consistent with mutational data as being important for the binding of Rev to the RRE. The G-G base pair may play an important role in opening the normally narrow major groove of A-form RNA to permit binding of the Rev basic domain.     

Saccharide-RNA recognition in a complex formed between neomycin B and an RNA aptamer.

BACKGROUND: Aminoglycoside antibiotics can target RNA folds with micromolar affinity and inhibit biological processes ranging from protein biosynthesis to ribozyme action and viral replication. Specific features of aminoglycoside antibiotic-RNA recognition have been probed using chemical, biochemical, spectroscopic and computational approaches on both natural RNA targets and RNA aptamers identified through in vitro selection. Our previous studies on tobramycin-RNA aptamer complexes are extended to neomycin B bound to its selected RNA aptamer with 100 nM affinity. RESULTS: The neamine moiety (rings I and II) of neomycin B is sandwiched between the major groove floor of a 'zippered-up' G.U mismatch aligned segment and a looped-out purine base that flaps over the bound antibiotic. Specific intermolecular hydrogen bonds are observed between the charged amines of neomycin B and base mismatch edges and backbone phosphates. These interactions anchor 2-deoxystreptamine ring I and pyranose ring II within the RNA-binding pocket. CONCLUSIONS: The RNA aptamer complexes with tobramycin and neomycin B utilize common architectural principles to generate RNA-binding pockets for the bound aminoglycoside antibiotics. In each case, the 2-deoxystreptamine ring I and an attached pyranose ring are encapsulated within the major groove binding pocket, which is lined with mismatch pairs. The bound antibiotic within the pocket is capped over by a looped-out base and anchored in place through intermolecular hydrogen bonds involving charged amine groups of the antibiotic.     

Structural basis of the highly efficient trapping of the HIV Tat protein by an RNA aptamer

An RNA aptamer containing two binding sites exhibits extremely high affinity to the HIV Tat protein. We have determined the structure of the aptamer complexed with two argininamide molecules. Two adjacent U:A:U base triples were formed, which widens the major groove to make space for the two argininamide molecules. The argininamide molecules bind to the G bases through hydrogen bonds. The binding is stabilized through stacking interactions. The structure of the aptamer complexed with a Tat-derived arginine-rich peptide was also characterized. It was suggested that the aptamer structure is similar for both complexes and that the aptamer interacts with two different arginine residues of the peptide simultaneously at the two binding sites, which could explain the high affinity to Tat.     

Rev-derived peptides inhibit HIV-1 replication by antagonism of Rev and a co-receptor,CXCR4

Rev, a viral regulatory protein of HIV-1, binds through its arginine-rich domain to the Rev-responsive element (RRE), a secondary structure in transcribed HIV-1 RNA. Binding of Rev to RRE mediates export of singly spliced or unspliced mRNAs from the nucleus to the cytoplasm. It has been previously shown that a certain arginine-rich peptide exhibits not only RRE-binding ability but also cell permeability and antagonism of CXCR4, one of the major coreceptors of HIV-1. Here we designed and synthesized arginine-rich peptides derived from the RNA-binding domain of Rev (Rev_34-50) and evaluated their anti-HIV-1 activities. Rev_34-50-A₄C, comprising Rev_34-50 with AAAAC at the C-terminus to increase the α-helicity, inhibited HIV-1 entry by CXCR4 antagonism and virus production in persistently HIV-1-infected PM1-CCR5 cells. Interestingly, similar motif of human lymphotropic virus type I Rex (Rex_1-21) also exerted moderate anti-HIV-1 activity. These results indicate that arginine-rich peptide, Rev_34-50-A₄C exerts dual antagonism against CXCR4 and Rev.