Rev Response Element RNA Research Articles

Evolvability of the mode of peptide binding by an RNA

The HIV Rev-response element (RRE) RNA binds strongly to two unrelated peptides, the HIV Rev peptide and an RRE-binding aptamer, the RSG-1.2 peptide, at a similar site, but using distinct sets of interactions. In this study, the nucleotide base requirements for the binding of the RRE to the Rev and RSG-1.2 peptides were determined by selection of Rev- and RSG-1.2-binding RRE variants using a bacterial reporter system. As a result, distinct differences in the bases necessary for binding the two peptides were found in the upper stem of the RRE. Strikingly, single nucleotide changes in this region were found to switch the peptide-binding specificity of the RRE from a bifunctional Rev- and RSG-1.2-binding mode to either a Rev-specific or a RSG-1.2- specific mode, demonstrating how an RNA can evolve alternative binding strategies in discrete steps without intermediate loss of function. This evolvability of the mode of peptide binding by an RNA presumably reflects the multidimensionality of conformational space that a given RNA has available for ligand recognition, which may have been utilized in the evolution of RNA-polypeptide complexes.

Method for Detection of Specific Nucleic Acids by Recombinant Protein with Fluorescent Resonance Energy Transfer

Detection of specific nucleic acids is important to understand cellular mechanisms and functions of gene regulation. Here, we demonstrated a novel method to detect specific nucleic acids using recombinant protein and oligonucleotides. A recombinant protein YRGnC-11ad, which has a Rev-peptide between enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) was constructed and expressed in HeLa cells. Rev-peptide, which corresponds to amino acids 34-50 of the HIV-1 Rev protein, indicates disordered structure in solution but forms alpha-helical and elongated conformation upon binding to Rev response element RNA (RRE-RNA) and Rev-aptamer, respectively. We confirmed that YRGnC-11ad could specifically bind to RRE-RNA and Rev-aptamer in cell lysate, and fluorescent resonance energy transfer (FRET) signal was changed upon binding following the conformational change of Rev-peptide. To utilize this FRET signal change toward the detection of specific nucleic acids, we split the RRE-RNA sequence and connected to the complementary oligonucleotide for target nucleic acids. When each two oligonucleotides hybridized to an adjacent region of target nucleic acids correctly, a Rev-peptide binding site was reformed on the hybridized complex. And we could confirm that YRGnC-11ad recombinant protein indicated FRET increase upon binding to the hybridized complex in cell lysate. These results suggest that the recombinant protein probe is available for specific nucleic acid detection.

Identification of mRNA that binds to eukaryotic initiation factor 5A by affinity co-purification and differential display.

Eukaryotic initiation factor 5A (eIF-5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genetic and pharmacological evidence suggests that eIF-5A is essential for cell survival and proliferation. However, the precise function and interacting partners of eIF-5A remain unclear. We have shown previously that eIF-5A can bind to RRE (Rev-response element) and U6 RNA in vitro. Using SELEX (systematic evolution of ligands by exponential enrichment), we have also shown that eIF-5A is capable of binding to RNA in a sequence-specific manner [Xu and Chen (2001) J. Biol. Chem. 276, 2555-2561]. In the present paper, we show that the identification of mRNA species that bind to eIF-5A can be achieved by affinity co-purification and PCR differential display. Using this approach with three sets of anchoring and arbitrary primers, we have found 20 RNA sequences that co-purified specifically with eIF-5A. Five of them contained AAAUGU, the putative eIF-5A-interacting element that we identified previously using the SELEX method. Direct binding of the cloned RNA to eIF-5A could be demonstrated by electrophoretic mobility-shift assay. BLAST analysis revealed that the eIF-5A-interacting RNAs encode proteins such as ribosomal L35a, plasminogen activation inhibitor mRNA-binding protein, NADH dehydrogenase subunit and ADP-ribose pyrophosphatase. Some, however, encode hypothetical proteins. All the cloned RNAs have the potential to form extensive stem-loop structures.

Library construction of neomycin–dipeptide heteroconjugates and selection against RRE RNA

An approach is described to the design of neomycin–dipeptide conjugates as ligands for Rev responsive element (RRE) RNA, which effectively inhibit Rev–RRE interaction. A library of 256 neomycin–dipeptide conjugates was constructed on TentaGel beads using a split-and-pool combinatorial synthesis. Five conjugates were selected after screening the library with fluorescence linked RRE RNA, and they were identified after sequencing by MALDI-TOF mass spectrometer. The heteroconjugates bind to RRE RNA with moderately improved affinities and highly improved specificity, compared to neomycin as determined by means of fluorescence anisotropy and surface plasmon resonance (SPR) experiments. This strategy, synthesis of the neomycin–peptide heteroconjugate library and selection against RNA target, could provide an efficient way to develop inhibitors against pathogenic RNA.

In Vivo HIV-1 Rev Multimerization in the Nucleolus and Cytoplasm Identified by Fluorescence Resonance Energy Transfer

Nuclear export of intron-containing human immunodeficiency virus type 1 RNA is mediated by the viral Rev protein. Rev is a nucleocytoplasmic transport protein that directly binds to its cis-acting Rev-responsive element RNA. Rev function depends on its ability to multimerize. The in vivo dynamics and the subcellular dependence of this process are still largely unexplored. To visualize and quantitatively analyze the mechanism of Rev multimeric assembly in live cells, we used high resolution in vivo fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching. By using two different dynamic FRET approaches (acceptor photobleaching and donor bleaching time measurements), we observed a strong Rev-Rev interaction in the nucleoli of living cells. Most interestingly, we could also detect Rev multimerization in the cytoplasm; however, FRET efficiency in the cytoplasm was significantly lower than in the nucleolus. By using fluorescence recovery after photobleaching, we investigated the mobility of Rev within the nucleolus. Mathematical modeling of the fluorescence recovery after photobleaching recoveries enabled us to extract relative association and dissociation constants and the diffusion coefficient of Rev in the nucleolus. Our results show that Rev multimerizes in the nucleolus of living cells, suggesting an important role of the nucleolus in nucleocytoplasmic transport.

Stereospecificity of short Rev-derived peptide interactions with RRE IIB RNA.

The essential HIV-1 regulatory protein Rev binds to the Rev responsive element (RRE) of the HIV-1 mRNA. A short alpha-helical peptide derived from Rev (Rev 34-50) and a truncated form of the RRE sequence (RRE IIB) provide a useful in vitro system to study the interactions between Rev and RRE. The current studies focus on evaluating the specificity of the binding interactions between Rev 34-50 and RRE IIB. The binding of L- and D-Rev peptides to natural and enantiomeric RRE IIB RNA was studied by fluorescence spectroscopy. D-Rev and L-Rev peptides bind to RRE IIB with similar affinities. CD measurements are consistent with a nonhelical, probably beta-hairpin, conformation for D-Rev in the complex. The binding affinities of D/L Rev peptides to L-RRE IIB RNA are also similar to those with natural D-RRE IIB. Furthermore, the conformations of L- and D-peptides when bound to L-RRE are reciprocal to the conformations of these peptides in complex with D-RRE. RNA footprinting studies show that L- and D-Rev peptides bind to the same site on RRE IIB. Our results demonstrate lack of stereospecificity in RRE RNA-Rev peptide interactions. However, it is quite possible that the interactions between full-length Rev protein and RRE are highly specific.

A nuclear kinesin-like protein interacts with and stimulates the activity of the leucine-rich nuclear export signal of the human immunodeficiency virus type 1 rev protein.

The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE). In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev/Rex effector binding protein), highly homologous to Kid, the carboxy-terminal 75-residue region of which interacts specifically with the NESs of HIV-1 Rev, human T-cell leukemia virus type 1 Rex, and equine infectious anemia virus Rev but not with functionally inactive mutants thereof. REBP is a nuclear protein that colocalizes with Rev in the nucleoplasm and nuclear periphery of transfected cells. Specific, albeit weak, interaction between REBP and Rev could be demonstrated in coimmunoprecipitation assays in BSC-40 cells. REBP can modestly enhance Rev-dependent RRE-linked reporter gene expression both independently and in cooperation with the nucleoporin cofactor Rab/hRIP. Thus, REBP displays the characteristics expected of an authentic mediator of Rev NES function and may play a role in RRE RNA transport during HIV infection.

Recruitment of the Crm1 nuclear export factor is sufficient to induce cytoplasmic expression of incompletely spliced human immunodeficiency virus mRNAs.

Cytoplasmic expression of the incompletely spliced RNA transcripts that encode the late, structural proteins of human immunodeficiency virus type 1 (HIV-1) is dependent on the viral Rev regulatory protein. General agreement exists that Rev acts, at least in part, by recruiting the cellular Crm1 nuclear export factor to HIV-1 transcripts bearing the Rev response element RNA target, and thereby inducing their nuclear egress. However, several groups have argued that Crm1 recruitment may not be sufficient for Rev function. Thus, several additional candidate cofactors for Rev have been proposed, and Rev has also been suggested to also inhibit the nuclear splicing of HIV-1 transcripts and/or to directly enhance their cytoplasmic translation. To examine whether Crm1 recruitment is, instead, sufficient to activate the nuclear export of viral mRNAs, we targeted a leucine-rich Crm1 binding domain, derived from a heterologous protein that normally plays no role in RNA metabolism, to HIV-1 RNAs and showed that this tethered Crm1 binding domain is sufficient to induce the nuclear export and cytoplasmic translation of late HIV-1 mRNA species. More importantly, we show that direct tethering of the Crm1 nuclear export factor to target mRNAs, by fusion to a heterologous RNA binding domain, is in and of itself sufficient to induce the nuclear export and cytoplasmic expression of the unspliced HIV-1 mRNAs that encode the viral Gag proteins.

Small molecule modulators of HIV Rev/Rev response element interaction identified by random screening

A high throughput scintillation proximity assay with biotinylated human immunodeficiency virus (HIV) Rev protein and tritiated Rev response element RNA was used to screen over 500 000 small molecules. Several chemical classes of inhibitors and two chemical classes of enhancers of binding were identified, with the molecular weight range being 400–600. The most common structural motif of inhibitor was an acidic moiety at the end of a linear aromatic system. Most of these modulators had EC 50 values in the 1–10 μM potency range, with several below 1 μM. Several classes displayed structure–activity relationships suggesting specific molecular interactions between small molecule and macromolecule. Several molecules were confirmed as inhibitors in a gel shift assay and by surface plasmon resonance analysis. Furthermore, one inhibitor was shown to bind the Rev protein with a binding constant equal to its IC 50 value, consistent with the mechanism of inhibition being binding Rev. Thus, small molecules can modulate this macromolecular protein–RNA interaction in vitro. However, no compound demonstrated HIV antiviral activity in a relevant cell-based assay.

Structural characterization of the complex of the Rev response element RNA with a selected peptide

Introduction: The RSG-1.2 peptide was selected for specific binding to the Rev response element RNA, as the natural Rev peptide does. The RSG-1.2 sequence has features incompatible with the helical structure of the bound Rev peptide, indicating that it must bind in a different conformation. Results: The binding of the RSG-1.2 peptide to the Rev response element RNA was characterized using multinuclear, multidimensional NMR. The RSG-1.2 peptide is shown to bind with the N-terminal segment of the peptide along the major groove in an extended conformation and turn preceding a C-terminal helical segment, which crosses the RNA groove in the region widened by the presence of purine–purine base pairs. These features make the details of the bound state rather different than that of the Rev peptide which targets the same RNA sequence binding as a single helix along the groove axis. Conclusions: These studies further demonstrate the versatility of arginine-rich peptides in recognition of specific RNA elements and the lack of conserved structural features in the bound state.

HIV Rev peptides conjugated with peptide nucleic acids and their efficient binding to RRE RNA

HIV Rev peptides conjugated with peptide nucleic acids (PNAs) were designed and synthesized to develop a designing approach for a novel RNA-binding molecule. The binding affinities of PNA-peptides with the Rev responsive element (RRE) RNA were determined by the competition assay using a rhodamine-labeled Rev. The peptide conjugated with an antisense PNA (TGCGC) bound RRE RNA more efficiently than the molecule without the PNA or the peptide sequence.

Binding of dimeric aminoglycosides to the HIV-1 rev responsive element (RRE) RNA construct

Through a series of elegant fluorescence measurements, particularly through stopped-flow kinetic measurements, it was recently demonstrated that aminoglycoside antibiotics are able to bind to the HIV-1 Rev responsive element (RRE) RNA construct in more than a 1:1 stoichiometry (Lacourciere, K. A.; Stivers, J. T.; Marino, J. P. Biochemistry 2000, 39, 5630). Here, we present the binding study results of dimeric neomycin ligands through fluorescence anisotropy studies, to the HIV-1 RRE RNA construct. The dimeric neomycin molecules are observed to be able to bind the HIV-1 RRE RNA construct approximately 17-fold higher when compared to the monomeric neomycin, lending evidence that there are indeed two or more neomycin binding sites within the HIV-1 RRE construct.

Exchange of the basic domain of human immunodeficiency virus type 1 Rev for a polyarginine stretch expands the RNA binding specificity, and a minimal arginine cluster is required for optimal RRE RNA binding affinity, nuclear accumulation, and trans-activation.

The Rev regulatory protein of human immunodeficiency virus (HIV) facilitates the nuclear export of unspliced and partially spliced HIV RNAs. Using a Rev:MS2 phage coat protein fusion that could be targeted to bind and activate the Rev-responsive element (RRE) RNA or heterologous MS2 phage operator RNA, we analyzed the role(s) of the arginine-rich RNA binding domain in RNA binding and transactivation. The arginine-rich domain could be functionally replaced by a stretch of nine arginines. However, polyarginine substitutions expanded the RNA binding specificity of the resultant mutant Rev protein. Polyarginine insertions in place of residues 24 to 60 that excised the RNA binding and oligomerization domains of Rev preserved the activation for MS2 RNA, but not for the RRE. A nine-arginine insertion outside of the natural context of the Rev nuclear localization signal domain was incompatible with activation of either RNA target. Insertions of fewer than eight arginines impaired RRE activation. Interrupted lysine clusters and disruption of the arginine stretch with lysine or neutral residues resulted in a similar phenotype. Some of these mutants with a null phenotype for RRE activated the heterologous MS2 RNA target. Under steady-state conditions, mutants that preserved the Rev response for RRE RNA localized to the nuclei; those with poor or no Rev response accumulated mostly in the cytoplasm. Many of the cytoplasmically resident derivatives became nuclear when leptomycin B (LMB) treatment inhibited nuclear export of nuclear export signal-containing proteins. Mutants that had a null activation potential for either RNA target were particularly resistant to LMB treatment. Abbreviated nuclear residence times and differences in RRE binding affinity may have compromised their activation potential for RRE. High-affinity binding to MS2 RNA through the intact coat protein was sufficient to overcome the short nuclear residence times and to facilitate MS2 activation by some derivatives.

1H-1H correlations across N-H...N hydrogen bonds in nucleic acids.

In 2HJ(NN)-COSY experiments, which correlate protons with donor/acceptor nitrogens across Nd...HNa bonds, the receptor nitrogen needs to be assigned in order to unambiguously identify the hydrogen bond. For many situations this is a non-trivial task which is further complicated by poor dispersion of (Na,Nd) resonances. To address these problems, we present pulse sequences to obtain direct, internucleotide correlations between protons in uniformly 13C/15N labeled nucleic acids containing Nd...HNa hydrogen bonds. Specifically, the pulse sequence H2(N1N3)H3 correlates H2(A,omega1):H3(U,omega2) protons across Watson-CrickA-U and mismatched G.A base pairs, the sequences H5(N3N1)H1/H6(N3N1)H1 correlate H5(C,omega1)/H6(C,omega1):H1(G,omega2) protons across Watson-Crick G-C base pairs, and the H2(N2N7)H8 sequence correlates NH2(G,A,C;omega1):H8(G,A;omega2) protons across G.G, A.A, sheared G.A and other mismatch pairs. These 1H-1H connectivities circumvent the need for independent assignment of the donor/acceptor nitrogen and related degeneracy issues associated with poorly dispersed nitrogen resonances. The methodology is demonstrated on uniformly 13C/15N labeled samples of (a) an RNA regulatory element involving the HIV-1 TAR RNA fragment, (b) a multi-stranded DNA architecture involving a G.(C-A) triad-containing G-quadruplex and (c) a peptide-RNA complex involving an evolved peptide bound to the HIV-1 Rev response element (RRE) RNA fragment.

Deletions near the N-terminus of HIV-1 Rev reduce RNA binding affinity and dominantly interfere with Rev function irrespective of the RNA target.

The contributions of the near N-terminal residues of Rev protein of HIV were investigated by analyzing N-terminal deletions of Rev in the context of a Rev/MS-C fusion protein that can bind and activate both the Rev responsive element (RRE) and the MS2 phage translational operator RNAs. Rev/MS-C fusion proteins deleted for residues 3-19 of Rev retained trans-activation potential for both RRE and MS2 targets. Coincidentally, peptides spanning residues 17-87 or 22-85 were functionally competent for trans-activation of RRE containing HIV-1 gag mRNA. Deletion of residues 18-24 of Rev in the Rev/MS-C fusion protein abolished the activation potential for both RRE and MS2 targets, although this mutant was competent for specific RNA binding, protein multimerization, and nuclear and nucleolar localization. Four mutants dominantly interfering with Rev activation of RRE were mapped near the N-terminus of Rev; (i) between residues 18 and 24, (ii) 25-34, (iii) 43-50, and (iv) 51-60. Of these, the mutant lacking residues 18-24 was a novel trans-dominant inhibitor of Rev and Rev/MS-C for activation of RRE or MS2 RNA, while the oligomerization domain mutants mapping between residues 25-34 or 51-60 inhibited the activation of RRE rather than MS2 RNA.

Construction of HIV Rev peptides containing peptide nucleic acid that bind HIV RRE IIB RNA

Peptides containing peptide nucleic acid (PNA) have been designed and synthesized to construct molecules recognizing a bulge or a loop structure of RNA. Such peptides were here designed from the HIV Rev protein that can bind the stem-loop IIB of the Rev responsive element (RRE) RNA. Variations of PNA modulated the binding affinities of the peptides to RRE IIB RNA.

A novel 5 displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA.

An RNA spin-labeling technique was developed using the well-characterized interaction between the HIV Rev peptide and the Rev response element (RRE) RNA as a model system. Spin-labeled RNA molecules were prepared by incorporating guanosine monophosphorothioate (GMPS) at the 5' end using T7 RNA polymerase and then covalently attaching a thiol-specific nitroxide spin label. Three different constructs of the RRE RNA were made by strategically displacing the 5' end within the native three-dimensional structure. Nitroxide-to-nitroxide distance measurements were made between the specifically bound RNA and peptide using electron paramagnetic resonance (EPR) spectroscopy. The dipolar EPR method can reliably measure distances up to 25 A, the calculation of which is derived from the 1/r3 dependence of the broadening of EPR lines in motionally frozen samples. This RNA-labeling technique, dubbed 5' displacement spin labeling, extends the usefulness of the dipolar EPR method developed for analysis of protein structure. The advantage of this technique is that it is applicable to large RNA systems such as the ribosome, which are difficult to study by other structural methods.

Real-time Kinetics of HIV-1 Rev-Rev Response Element Interactions: DEFINITION OF MINIMAL BINDING SITES ON RNA AND PROTEIN AND STOICHIOMETRIC ANALYSIS

The kinetics of interaction between the human immunodeficiency virus-1 Rev protein and its RNA target, Rev response element (RRE) RNA was determined in vitro using a biosensor technique. Our results showed that the primary Rev binding site is a core stem-loop RNA molecule of 30 nucleotides that bound Rev at a 1:1 ratio, whereas the 244-nucleotide full-length RRE bound four Rev monomers. At high Rev concentrations, additional binding of Rev to RRE was observed with ratios of more than 10:1. Because RRE mutants that lacked the core binding site and were inactive in vivo bound Rev nonspecifically at these concentrations, the real stoichiometric ratio of Rev-RRE is probably closer to 4:1. Binding affinity of Rev for RRE was approximately 10(-10) M, whereas the affinity for the core RNA was about 10(-11) M, the difference being due to the contribution of low affinity binding sites on the RRE. Mathematical analysis suggested cooperativity of Rev binding, probably mediated by the Rev oligomerization domains. C-terminal deletions of Rev had no effect on RRE binding, but truncation of the N terminus by as few as 11 residues significantly reduced binding specificity. This method was also useful to rapidly evaluate the potential of aminoglycoside antibiotics, to inhibit the Rev-RRE interaction.

Analysis of Intracellular Trafficking and Interactions of Cytoplasmic HIV-1 Rev Mutants in Living Cells

The HIV-1 Rev protein is an essential nuclear regulatory viral protein. Rev mutants that are able to block wild-type (WT) Rev activityin transhave been reported and used in antiviral approaches. Not only nuclear but also cytoplasmic Rev mutants were described and suspected to be transdominant by retaining WT Rev in the cytoplasm. To investigate their potential for cytoplasmic retention, we studied the localization, trafficking, and interactions of cytoplasmic Rev mutants containing mutations in the N-terminal multifunctional domain. Using a novel dual-color autofluorescent protein-tagging system, we found that coexpression of the nucleolar blue-tagged WT Rev protein together with green-labeled cytoplasmic Rev mutants did not result in the retention of WT Rev in the cytoplasm but, on the contrary, in colocalization of the mutants to the nucleolus. A combination of mutations abolished the interaction with WT Rev, defining two domains important for Rev protein interaction. The identified domains were also essential for specific Rev responsive element (RRE) RNA binding and nuclear retention. Inactivation of the nuclear export signal shifted the steady-state distribution of the mutants from the cytoplasm to the nucleus, indicating their capability for nucleo-cytoplasmic shuttling. The cytoplasmic mutants were not transdominant compared to the nuclear mutant RevM10BL. These results emphasize that efficient oligomerization with WT Rev combined with RRE-specific RNA binding are prerequisites for effective transdominance.

Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1.

The hypothesis that the cellular protein Crm1 mediates human immunodeficiency virus type 1 (HIV-1) Rev-dependent nuclear export posits that Crm1 can directly interact both with the Rev nuclear export signal (NES) and with cellular nucleoporins. Here, we demonstrate that Crm1 is indeed able to interact with active but not defective forms of the HIV-1 Rev NES and of NESs found in other retroviral nuclear export factors. In addition, we demonstrate that Crm1 can bind the Rev NES when Rev is assembled onto the Rev response element RNA target and that Crm1, like Rev, is a nucleocytoplasmic shuttle protein. Crm1 also specifically binds the Rev NES in vitro, although this latter interaction is detectable only in the presence of added Ran . GTP. Overexpression of a truncated, defective form of the nucleoporin Nup214/CAN, termed DeltaCAN, that retains Crm1 binding ability resulted in the effective inhibition of HIV-1 Rev or human T-cell leukemia virus Rex-dependent gene expression. In contrast, DeltaCAN had no significant affect on Mason-Pfizer monkey virus constitutive transport element (MPMV CTE)-dependent nuclear RNA export or on the expression of RNAs dependent on the cellular mRNA export pathway. As a result, DeltaCAN specifically blocked late, but not early, HIV-1 gene expression in HIV-1-infected cells. These data strongly validate Crm1 as a cellular cofactor for HIV-1 Rev and demonstrate that the MPMV CTE nuclear RNA export pathway uses a distinct, Crm1-independent mechanism. In addition, these data identify a novel and highly potent inhibitor of leucine-rich NES-dependent nuclear export.