Wild-type Rev Research Articles

Altered‐specificity mutants of the HIV Rev arginine‐rich motif‐RRE IIB interaction

Arginine-rich motifs (ARMs) bind RNA structures with high affinity and specificity, and the human immunodeficiency virus (HIV) exploits ARM-RNA interactions to regulate its lifecycle. The expression of HIV structural genes relies on recognition between the ARM of its Rev protein and its primary binding site, an internal loop in the viral RNA, the Rev-response element region IIB (IIB). Many functional variants of the Rev ARM-IIB interaction have been discovered, yet how easily it can evolve new specificities is poorly explored. A double mutant of Rev ARM, R35G-N40 V, uses an unknown strategy to recognize IIB. Here, isothermal titration calorimetry and gel shift assays show that the R35G-N40V-IIB interaction has high affinity and specificity in vitro and a larger unfavorable entropy change upon binding than that of wild-type Rev ARM-IIB. In stark contrast with the critical dependence of wild-type Rev on Arg35, Arg39, Asn40, and Arg44, mutational profiling shows R35G-N40V is highly mutable at positions 40 and 44 and dependent on Gly35, Arg38, Arg39, Arg42, and Arg43. Affinity measurements in vitro and reporter assay measurements in vivo are consistent with the wild-type Rev ARM and R35G-N40V maintaining their recognition strategies when binding IIB mutants specific to wild-type Rev ARM and R35G-N40V, respectively. Some single amino acid mutants of wild-type Rev ARM and R35G-N40V have enhanced specificity, recognizing mutant IIBs yet not wild-type IIB. These results provide another example of viral ARM-RNA interactions evolving new specificities with few mutations, consistent with neutral theories of evolution.

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry.

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

Diverse mutants of HIV RRE IIB recognize wild-type Rev ARM or Rev ARM R35G-N40V.

The binding of human immunodeficiency virus Rev protein via its arginine-rich motif (ARM) to an internal loop in the Rev-response element region IIB (RRE IIB) is necessary for viral replication. Many variant RNAs and ARMs that bind Rev and RRE IIB have been found. Despite the essential role of Rev asparagine 40 in recognition, the Rev ARM double-mutant R35G-N40V functions well in a Rev-RRE IIB reporter assay, indicating R35G-N40V uses a distinct recognition strategy. To examine how RRE IIB may evolve specificity to wild-type Rev ARM and R35G-N40V, 10 RRE IIB libraries, each completely randomized in overlapping regions, were screened with wild-type Rev ARM and R35G-N40V using a reporter system based on bacteriophage λ N antitermination. Consistent with previous studies, a core element of RRE IIB did not vary, and substitutions occurred at conserved residues only in the presence of other substitutions. Notably, the groove-widening, non-canonical base-pair G48:G71 was mutable to U48:G71 without strong loss of binding to wild-type Rev ARM, suggesting U48:G71 performs the same role by adopting the nearly isosteric, reverse wobble base pair. Originating from RRE IIB, as few as one or two substitutions are sufficient to confer specificity to wild-type Rev or Rev R35G-N40. The diversity of RRE IIB mutants that maintain binding to wild-type Rev ARM and R35G-N40V supports neutral theories of evolution and illustrates paths by which viral RNA-protein interactions can evolve new specificities. Rev-RRE offers an excellent model with which to study the fine structure of how specificity evolves.

Over-expression of the HIV-1 Rev promotes death of nondividing eukaryotic cells

Expression of the human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for completion of the viral life cycle. Rev mediates nuclear export of partially spliced and unspliced viral transcripts and therefore bears a nuclear localization signal (NLS) as well as a nuclear export signal (NES), which allow its nucleocytoplasmic shuttling. Attempts to express the wild-type Rev protein in eukaryotic human cultured cells have encountered difficulties and so far have failed. Here we show that accumulation of Rev, which occurs in nondividing Rev-expressing cells or when such cells reach confluency, results in death of these cells. Cell death was also promoted by addition of a cell permeable peptide bearing the Rev-NES sequence, but not by the Rev-NLS peptide. Our results probably indicate that binding of excess amounts of the Rev protein or the NES peptide to the exportin receptor CRM1 results in cells' death.

Functional rescue of an oligomerization-defective HIV-1 Rev mutant by fusion with an oligomeric tag

The HIV-1 wild-type Rev and the negative oligomerization-defective mutant RevM4 were fused to enhanced green fluorescent protein (EGFP) and the tetrameric red fluorescent protein (DsRed1) followed by examination of their intracellular localization and Rev activity. As previously shown, fusion of EGFP to Rev and RevM4 did not affect the biological activity. Fusion of DsRed1 to Rev inhibited activity, and interestingly, fusion of DsRed1 to RevM4 restored activity. Based on these results, a model is proposed suggesting how RevM4-DsRed1 is able to rescue Rev activity through oligomerization on the viral RNA.

An α-Helical Peptidomimetic Inhibitor of the HIV-1 Rev−RRE Interaction

The interaction between the HIV-1 Rev protein and the Rev-Responsive Element (RRE) RNA is an attractive target for anti-viral therapy. We have designed alpha-helical peptidomimetics of Rev-like peptides using side chain-side chain macrolactam formation between positions i and i+4. One peptidomimetic having an appropriate location, orientation, and length of the macrolactam exhibited both significant helical character and specific RRE binding. This molecule displays 2-fold greater RNA specificity than the wild-type Rev peptide and more than 20-fold greater specificity than an uncyclized control peptide. Thus, specific, high affinity recognition of the RRE is feasible utilizing a small, relatively rigid peptidomimetic scaffold.

Phenotypic analysis of human immunodeficiency virus type 1 Rev trimerization-interface mutants in human cells

Nuclear export of unspliced and incompletely spliced human immunodeficiency virus type 1 mRNA is mediated by the viral Rev protein. Rev binds to a structured RNA motif known as the Rev-response element (RRE), which is present in all Rev-dependent transcripts, and thereby promotes entry of the ribonucleoprotein complex into the nuclear-export pathway. Recent evidence indicates that a dimerization interface and a genetically separable "trimerization" interface are required for multimeric assembly of Rev on the RRE. In this report, the effect of mutations within the trimerization interface on Rev function was examined in mammalian cells. All trimerization-defective Rev molecules had profoundly compromised Rev function and a range of localization defects was observed. However, despite the potential for formation of heterodimers between functional and non-functional Rev proteins, trimerization-defective Rev mutants were unable to inhibit wild-type Rev function in a trans-dominant-negative manner.

Restriction of Human Immunodeficiency Virus Type 1 Rev Function in Murine A9 Cells Involves the Rev C-Terminal Domain

The human immunodeficiency virus type 1 (HIV-1) Rev and human T-cell leukemia virus type 1 (HTLV-1) Rex proteins are essential for the expression of viral structural proteins and productive infection. Both contain a nuclear export signal (NES) in their C-terminal domain and a nuclear localization signal (NLS) in their N-terminal domain. The NES and NLS are necessary for shuttling between nucleus and cytoplasm and are therefore indispensable for the transport of unspliced and singly spliced viral transcripts. HIV-1 Rev function is restricted in A9 cells, a murine fibroblast cell line, whereas HTLV-1 Rex is functional in these cells. Immunofluorescence studies with RevGFP fusion protein demonstrate normal import and export of Rev in A9 cells. To ascertain which domains of Rev are necessary for the restriction of Rev function in A9 cells, we studied a chimeric construct in which the NES domain of Rev was exchanged with Rex C-terminal amino acids 79 to 95, the Rev1-79/Rex79-95 chimera, which restored Rev function in A9 cells. In addition, overexpression of a truncated Rev containing the Rev C-terminal domain in the presence of wild-type Rev, led to restoration of Rev function in A9 cells. These results suggest that the C-terminal domain of HIV-1 Rev plays an important role in restricting Rev function in murine cells.

A trans-dominant negative HIV type 1 Rev with intact domains of NLS/NOS and NES.

The nuclear diffusion inhibitory signal (NIS) is a 15-amino acid peptide motif (10-24; EDLLKAVRLIKFLYQ) in the N-terminus of the HIV-1 Rev protein. NIS appears to be involved in maintaining the proper nucleocytoplasmic trafficking and intracellular stability of HIV-1 Rev. Deletion in NIS leads to Rev functional inactivity, and these data led to further investigation of its possible inhibitory effects on Rev function. An HIV-1 proviral rescue assay was utilized to evaluate Rev function. The association between wild-type Rev molecules, or wild-type Rev with Revd23, an NIS mutant, plus Rev-responsive element (RRE) interactions in cultured cells were also evaluated. Revd23 showed a potent trans-dominant negative phenotype, while multimerization with wild-type Rev and Revd23-RRE binding in cells were found to reveal no significant changes from wildtype. These results suggest that the potential trans-dominance mechanism of Revd23 may differ from that of a Rev construct, RevM10, with mutations in the C-terminus nuclear export signal (NES). As such, these data will be useful in the rational design of novel antiretroviral approaches targeting HIV-1 Rev.

Anti-human immunodeficiency virus type 1 gene therapy in human central nervous system-based cells: an initial approach against a potential viral reservoir.

Studies have demonstrated that human immunodeficiency virus type 1 (HIV-1) infection of central nervous system (CNS)-based cells in vivo results in a series of devastating clinical conditions collectively termed acquired immune deficiency syndrome (AIDS) dementia complex (ADC). Gene therapy for these neurovirological disorders necessitates utilization of a vector system that can mediate in vivo delivery and long-term expression of an antiretroviral transgene in nondividing/postmitotic CNS cellular elements. The present studies focus on the transfer of an anti-HIV-1 gene to primary isolated CNS microvascular endothelial cells (MVECs) and neuronal-based cells, for its effects in protecting these cells from HIV-1 infection. By using an HIV-1-based vector system, it was possible to efficiently transduce and maintain expression of a marker transgene, beta-galactosidase (beta-Gal), in human CNS MVECs, human fetal astrocytes, plus immature and mature (differentiated) NT2 cells. Significant transduction of the marker gene, beta-Gal, in CNS-based cells prompted the utilization of this system with an anti-HIV-1 gene therapeutic construct, RevM10, a trans-dominant negative mutant Rev protein. Initially, it was not possible to generate any HIV-1 vector particles with the RevM10 gene in the transducing construct, because of inhibitory effects on the HIV-1 vector by this gene product. However, the vector could be partially rescued by adding an additional construct that supplied wild-type rev, in trans, during a multiple construct transfection in the packaging 293T cells. Thus, it was possible to significantly improve the titer of RevM10-expressing viral particles generated from these cells. Moreover, this RevM10 vector transduced the neuronal precursor cell line NT2, retinoic acid-differentiated human neurons (hNT) from the precursor cells, and primary isolated human brain MVECs with high efficiency. RevM10 generated from the HIV-1-based vector system potently inhibited replication of diverse HIV-1 strains in human CNS MVECs and neuronal cells. The data generated from these studies represent an initial approach for future development of anti-HIV-1 gene therapy in the CNS.

In vivo selection of RNA-binding peptides from combinatorial libraries.

We have used a two step procedure to identify peptides that bind strongly to the Rev-response element (RRE) of HIV. In the first step, RRE-binding peptides were screened from a combinatorial peptide library generated by "randomization" using a small subset of the 20 amino acids. In the second step, one such RRE-binding peptide, RSG-1, was "evolved" into an even stronger RRE-binding peptide using a codon-based mutagenesis procedure. After 2 rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity than wild-type Rev peptide.

Co-expression of a trans-dominant negative mutant of the human immunodeficiency virus type 1 (HIV-1) Rev protein affects the Rev-dependent splicing pattern and expression of HIV-1 RNAs.

Trans-dominant negative mutants of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev inhibit the function of wild-type Rev in a dose-dependent manner. This was previously shown to be caused by nuclear retention of the wild-type protein. In the present work, further analysis of the trans-dominant negative effect was performed using cotransfection experiments with different constructs encoding HIV-1 Rev and viral structural proteins together with a plasmid encoding a trans-dominant negative Rev mutant. Thus, one species of pre-mRNA was transcribed from the reporter plasmids. This pre-mRNA was then either spliced or exported by Rev as unspliced RNA for translation of the HIV structural proteins. An immunofluorescence assay and Western blot analysis were used for analysis of protein expression. In situ hybridization was applied for labelling of unspliced mRNA in transfected cells, and RNase protection analysis was used to determine the relative amount of unspliced versus spliced mRNAs. The experiments confirmed that the transdominant negative mutant inhibited nuclear export of unspliced mRNA. It was, in addition, demonstrated for the first time that the trans-dominant negative mutant also affected a Rev-dependent regulatory step connected with viral pre-mRNA splicing. As a consequence, proteins expressed from unspliced and singly spliced HIV mRNAs decreased while there was an increase in protein products encoded by spliced and alternatively spliced mRNAs.

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.

Chimeras Containing Influenza NS1 and HIV-1 Rev Protein Sequences: Mechanism of Their Inhibition of Nuclear Export of Rev Protein–RNA Complexes

Nuclear RNA export mediated by the HIV-1 Rev protein is inhibited by chimeric proteins in which the wild-type Rev protein is covalently linked to amino acid sequences of the NS1 protein of influenza A virus (NS1A protein), a protein that inhibits nuclear RNA export. These chimeric molecules function not only incisbut also intrans:they inhibit nuclear RNA export mediated by Rev protein molecules that are not covalently linked to the NS1A protein sequence. Here we show that inhibition occurs with a NS1-Rev chimera in which the 78 amino-terminal amino acids of the NS1A protein comprising its entire RNA-binding domain is deleted, thereby establishing that this carboxyl portion of the NS1A protein can function as an independent effector domain. The mechanism by which this NS1-Rev chimera inhibits Rev function intranswas determined. The Rev sequence in this chimera oligomerizes with Rev molecules intrans,and the resulting mixed oligomers are retained in the nucleus because the nuclear retention activity of the NS1 effector domain is dominant over the nuclear transport activity of the Rev effector domain. Binding of the FG-containing nucleoporin-like Rab protein to this NS1-Rev chimera, as measured in yeast two-hybrid assays, is much stronger than that to the Rev protein itself, yet nuclear export does not occur in the presence of the chimera. Unexpectedly, the introduction of specific mutations into the NS1A portion of this NS1-Rev chimera not only restores Rev-mediated nuclear export of RNA but also eliminates detectable Rab binding, indicating that this nuclear export can occur without detectable Rab binding. A different NS1-Rev chimera, one in which the NS1A protein is full-length but contains a mutated RNA-binding domain, effectively inhibits Rev-mediated nuclear export of RNA without blocking the nuclear export of the Rev protein, indicating that nuclear export of the carrier Rev protein can be uncoupled from nuclear export of its passenger RNA.

Structural dynamics of HIV-1 Rev and its complexes with RRE and 5S RNA.

The Rev protein of the human immunodeficiency virus type 1 (HIV-1) has been studied by time-resolved fluorescence spectroscopy. The single tryptophan residue of Rev, Trp45, located within the arginine-rich RNA-binding domain of the protein, was utilized as an intrinsic spectroscopic probe. In addition, five peptides spanning different lengths of the arginine-rich domain, each containing the tryptophan residue, and two C-terminal deletion mutants of Rev, Rev M9 delta 14 and Rev M11 delta 14, were examined. Rev M9 delta 14 lacks residues 68-112 whereas Rev M11 delta 14 is missing residues 92-112 of the C-terminus of Rev. The fluorescence decay of Trp45 in wild-type Rev was resolved into four discrete lifetime components, and decay-associated spectra (DAS) were obtained for each component. The fluorescence decays of all five peptides and Rev M9 delta 14 were resolved into three lifetime components. The fluorescence decay of Rev M11 delta 14 was resolved into four components similar to those found for wild-type Rev. These results indicate that the activation domain (residues 78-93), present in wild-type Rev and Rev M11 delta 14, induced a unique tryptophan environment, characterized by a short-lived, blue-shifted emission, attributed to higher order assembly of Rev. In addition, fluorescence anisotropy decay data obtained for wild-type Rev and the two C-terminal deletion mutants also indicate that the activation domain mediates self-association of Rev. Based on the anisotropy decay results for wild-type Rev, the distribution of oligomers is independent of salt concentration. The average fluorescence lifetime of Trp45 was reduced upon complexation of Rev with a 40-mer fragment of the Rev response element containing the minimal element for Rev binding (F8-RRE), and the emission was blue-shifted. In addition, the local rotation of the tryptophan side chain was blocked in the protein-RRE complex. These results indicate that Trp45 directly interacts with the RRE. Rev is also shown to bind to 5S RNA, resulting in very similar changes in the time-resolved tryptophan fluorescence to those observed upon complexation of Rev with F8-RRE.

A naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production.

We have isolated a naturally arising human immunodeficiency type 1 (HIV-1) mutant containing a point mutation within the env gene. The point mutation resulted in complete loss of balanced splicing, with dominant production of aberrant mRNAs. The aberrant RNAs arose via activation of normally cryptic splice sites flanking the mutation within the env terminal exon to create exon 6D, which was subsequently incorporated in aberrant env, tat, rev, and nef mRNAs. Aberrant multiply spliced messages contributed to reduced virus replication as a result of a reduction in wild-type Rev protein. The point mutation within exon 6D activated exon 6D inclusion when the exon and its flanking splice sites were transferred to a heterologous minigene. Introduction of the point mutation into an otherwise wild-type HIV-1 proviral clone resulted in virus that was severely inhibited for replication in T cells and displayed elevated usage of exon 6D. Exon 6D contains a bipartite element similar to that seen in tat exon 3 of HIV-1, consisting of a potential exon splicing silencer (ESS) juxtaposed to a purine-rich sequence similar to known exon splicing enhancers. In the absence of a flanking 5' splice site, the point mutation within the exon 6D ESS-like element strongly activated env splicing, suggesting that the putative ESS plays a natural role in limiting the level of env splicing. We propose, therefore, that exon silencers may be a common element in the HIV-1 genome used to create balanced splicing of multiple products from a single precursor RNA.

Molding a peptide into an RNA site by in vivo peptide evolution.

Short peptides corresponding to the arginine-rich domains of several RNA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities. In the case of the HIV-1 Rev-Rev response element (RRE) complex, the peptide forms a single alpha-helix that binds deeply in a widened, distorted RNA major groove and makes a substantial set of base-specific and backbone contacts. Using a reporter system based on antitermination by the bacteriophage lambda N protein, it has been possible to identify novel arginine-rich peptides from combinatorial libraries that recognize the RRE with affinities and specificities similar to Rev but that appear to bind in nonhelical conformations. Here we have used codon-based mutagenesis to evolve one of these peptides, RSG-1, into an even tighter binder. After two rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity and 15-fold higher specificity than the wild-type Rev peptide, and in vitro competition experiments show that RSG-1.2 completely displaces the intact Rev protein from the RRE at low peptide concentrations. By fusing RRE-binding peptides to the activation domain of HIV-1 Tat, we show that the peptides can deliver Tat to the RRE site and activate transcription in mammalian cells, and more importantly, that the fusion proteins can inhibit the activity of Rev in chloramphenicol acetyltransferase reporter assays. The evolved peptides contain proline and glutamic acid mutations near the middle of their sequences and, despite the presence of a proline, show partial alpha-helix formation in the absence of RNA. These directed evolution experiments illustrate how readily complex peptide structures can be evolved within the context of an RNA framework, perhaps reflecting how early protein structures evolved in an "RNA world."

A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase.

. The subcellular location of several nonribosomal nucleolar proteins was examined at various stages of mitosis in synchronized mammalian cell lines including HeLa, 3T3, COS-7 and HIV-1 Rev-expressing CMT3 cells. Nucleolar proteins B23, fibrillarin, nucleolin and p52 as well as U3 snoRNA were located partially in the peripheral regions of chromosomes from prometaphase to early telophase. However, these proteins were also found in large cytoplasmic particles, 1-2 microm in diameter, termed nucleolus-derived foci (NDF). The NDF reached maximum numbers (as many as 100 per cell) during mid- to late anaphase, after which their number declined to a few or none during late telophase. The decline in the number of NDF approximately coincided with the appearance of prenucleolar bodies and reforming nucleoli. The HIV-1 Rev protein and a mutant Rev protein defective in its nuclear export signal were also found in the NDF. The mutant Rev protein precisely followed the pattern of localization of the above nucleolar proteins, whereas the wild-type Rev did not enter nuclei until G1 phase. The nucleolar shuttling phosphoprotein Nopp140 did not follow the above pattern of localization during mitosis: it dispersed in the cytoplasm from prometaphase through early telophase and was not found in the NDF. Although the NDF and mitotic coiled bodies disappeared from the cytoplasm at approximately the same time during mitosis, protein B23 was not found in mitotic coiled bodies, nor was p80 coilin present in the NDF. These results suggest that a class of proteins involved in preribosomal RNA processing associate with chromosome periphery and with NDF as part of a system to conserve and deliver preexisting components to reforming nucleoli during mitosis.

A dynamic in Vivo view of the HIV-IRev-RRE interaction

The export of pre-mRNAs coding for the structural genes of the human immunodeficiency virus type I depends on the interaction of the Rev protein with a highly structured viral RNA sequence, the Rev-responsive element (RRE). To gain information about the structure of the RRE and the determinants of the in vivo RRE-Rev interaction, we have analyzed the structure of the 351 nt RRE RNA within living yeast (Saccharomyces cerevisiae) by dimethyl sulfate probing with or without Rev. The in vivo structure in the absence of Rev is generally similar to the previously established solution structure. In addition, we observe a single hypermethylated guanine residue (G128), located within the Rev high-affinity binding site, in vitro as well as in vivo. The important homopurine interaction between residues 129 and 106 is required for the hyperreactivity, confirming its biological relevance. Expression of wild-type Rev leads to a protection of this region and to modifications of the RRE structure: the high-affinity site becomes further structured, and Stem IIA is destabilized. High-level expression of the oligomerization-defective mutant M4 protein leads to the same protections without destabilization of Stem IIA. Taken together with other observations, the data suggest that Rev captures the unusual conformation of the high-affinity site, followed by additional changes in the structure of the RRE.

Structural Change in Rev Responsive Element RNA of HIV-1 on Binding Rev Peptide

The HIV-1 Rev responsive element (RRE) high-affinity binding site was studied by homonuclear and heteronuclear NMR. Two Rev binding element (RBE) RNA oligonucleotides were used as model systems in this study: RBE3, which contains the wild-type Rev high-affinity binding site, and RBE3-A which is identical except for the deletion of a bulged A. The temperature dependence of the two-dimensional spectra of the free RNAs indicates that at lower temperatures more than one conformation is present. However, at higher temperatures a single conformation predominates. Model structures of RBE3 and RBE3-A as well as the RBE3-A complexed with a peptide derived from the RNA binding domain of HIV-1 Rev, were calculated using NMR-derived restraints. The Rev high-affinity binding site of the HIV-1 RRE contains a structured internal loop with two purine·purine base-pairs and an extrahelical U. Comparison of the free and bound RNA structures reveals that upon peptide binding there is a distinct change in the backbone at G24, which is involved in a G·G base-pair. In the free RNA, G24 is in the synconformation, and the backbone is in a relatively normal configuration, antiparallel to the other strand. In the bound RNA, the backbone at G24 has flipped over so that it is parallel to the other strand. G24 in the bound RNA still forms a base-pair with G6, but is now in the anticonformation.