Recognition of the high affinity binding site in rev-response element RNA by the human immunodeficiency virus type-1 rev protein.

The Human Immunodeficiency Virus type-1 rev protein binds with high affinity to a bubble structure located within the rev-response element (RRE) RNA in stemloop II. After this initial interaction, additional rev molecules bind to the RRE RNA in an ordered assembly process which requires a functional bubble structure, since mutations in the bubble sequence that reduce rev affinity block multiple complex formation. We have used synthetic chemistry to characterize the interaction between rev protein and its high affinity binding site. A minimal synthetic duplex RNA (RBC6) carrying the bubble and 12 flanking base pairs is able to bind rev with 1 to 1 stoichiometry and with high affinity. When the bubble structure is inserted into synthetic RNA molecules carrying longer stretches of flanking double-stranded RNA, rev forms additional complexes resembling the multimers observed with the RRE RNA. The ability of rev to bind to RBC6 analogues containing functional group modifications on base and sugar moieties of nucleoside residues was also examined. The results provide strong evidence that the bubble structure contains specific configurations of non-Watson--Crick G:G and G:A base pairs and suggest that high affinity recognition of RRE RNA by rev requires hydrogen bonding to functional groups in the major groove of a distorted RNA structure.     

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. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification and characterization of a nuclear factor that specifically binds to the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1)

The Rev protein of human immunodeficiency virus type 1 (HIV-1) differentially transactivates the expression of viral structural proteins by allowing the accumulation of unspliced and singly spliced viral mRNA in the cytoplasm. The cis-acting RNA target sequence for the Rev protein, termed the Rev response element (RRE), is present in the env gene and is predicted to form a highly ordered RNA secondary structure. Recent data indicate that Rev directly binds to RRE and, further, that this binding can be mapped to a 90-nucleotide subfragment at the 5' end of RRE. We now report that RRE also binds specifically and predominantly to a nuclear factor of approximately 56 kD. Mapping of the binding site reveals that the same subfragment that binds Rev also binds this nuclear factor. We designate this protein as NFRRE for nuclear factor, RRE binding. Rev and NFRRE appear to bind simultaneously to RRE. NFRRE is widely distributed in various mammalian cells. We speculate that this factor plays an important role in Rev-mediated transactivation and is likely to be involved in the processing or transport of cellular mRNA.     

Molecular recognition of a branched peptide with HIV-1 Rev Response Element (RRE) RNA

Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein, Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a K_d value of 410?nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites.     

Water, Shape Recognition, Salt Bridges, and Cation-Pi Interactions Differentiate Peptide Recognition of the HIV Rev-Responsive Element

Recognition of the human immunodeficiency virus Rev-responsive element (RRE) RNA by the Rev protein is an essential step in the viral life cycle. Formation of the Rev-RRE complex signals nucleocytoplasmic export of unspliced and partially spliced viral RNA. Essential components of the complex have been localized to a minimal arginine-rich Rev peptide and stem IIB of RRE. In vitro selection studies have identified a synthetic peptide known as RSG 1.2 that binds with better specificity and affinity to RRE than the Rev peptide. NMR structures of both peptide-RNA complexes of Rev and RSG 1.2 bound to RRE stem IIB have been solved and reveal gross structural differences between the two bound complexes. Molecular dynamics simulations of the Rev and RSG 1.2 peptides in complex with RRE stem IIB have been simulated to better understand on an atomic level how two arginine-rich peptides of similar length recognize the same sequence of RNA with such different structural motifs. While the Rev peptide employs some base-specific hydrogen bonding for recognition of RRE, shape recognition, through contact with the sugar-phosphate backbone, and cation-pi interactions are also important. Molecular dynamics simulations suggest that RSG 1.2 binds more tightly to the RRE sequence than Rev by forming more base-specific contacts, using water to mediate peptide-RNA contacts, and is held in place by a strong salt bridge network spanning the major groove of the RNA.     

Fluorescence-based methods for evaluating the RNA affinity and specificity of HIV-1 Rev-RRE inhibitors

RNA plays a pivotal role in the replication of all organisms, including viral and bacterial pathogens. The development of small molecules that selectively interfere with undesired RNA activity is a promising new direction for drug design. Currently, there are no anti-HIV treatments that target nucleic acids. This article presents the HIV-1 Rev response element (RRE) as an important focus for the development of antiviral agents that target RNA. The Rev binding site on the RRE is highly conserved, even between different groups of HIV-1 isolates. Compounds that inhibit HIV replication by binding to the RRE and displacing Rev are therefore expected to retain activity across groups of genetically diverse HIV infections. Systematic evaluations of both the RRE affinity and specificity of numerous small molecule inhibitors are essential for deciphering the parameters that govern effective RRE recognition. This article discusses fluorescence-based techniques that are useful for probing a small molecule's RRE affinity and its ability to inhibit Rev-RRE binding. Rev displacement experiments can be conducted by observing the fluorescence anisotropy of a fluorescein-labeled Rev peptide, or by quantifying its displacement from a solid-phase immobilized RRE. Experiments conducted in the presence of competing nucleic acids are useful for evaluating the RRE specificity of Rev-RRE inhibitors. The discovery and characterization of new RRE ligands are described. Eilatin is a polycyclic aromatic heterocycle that has at least one binding site on the RRE (apparent Kd is approximately 0.13 microM), but it does not displace Rev upon binding the RRE (IC50 > 3 microM). In contrast, ethidium bromide and two eilatin-containing metal complexes show better consistency between their RRE affinity and their ability to displace a fluorescent Rev peptide from the RRE. These results highlight the importance of conducting orthogonal binding assays that establish both the RNA affinity of a small molecule and its ability to inhibit the function of the RNA target. Some Rev-RRE inhibitors, including ethidium bromide, Lambda-[Ru(bpy)(2)eilatin]2+, and Delta-[Ru(bpy)(2)eilatin]2+ also inhibit HIV-1 gene expression in cell cultures (IC50 = 0.2-3 microM). These (and similar) results should facilitate the future discovery and implementation of anti-HIV drugs that are targeted to viral RNA sites. In addition, a deeper general understanding of RNA-small molecule recognition will assist in the effective targeting of other therapeutically important RNA sites.     

Site-specific crosslinks of yeast U6 snRNA to the pre-mRNA near the 5' splice site.

We have introduced a single photochemical crosslinking reagent into specific sites in the central domain of U6 to identify the sites that are in close proximity to the pre-mRNA substrate. Four distinct U6 snRNAs were synthesized with a single 4-thiouridine (4-thioU) at positions 46, 51, 54, and 57, respectively. Synthetic U6 RNA containing the 4-thioU modifications can functionally reconstitute splicing activity in cell-free yeast splicing extracts depleted of endogenous U6 snRNA. Upon photoactivation with UV (>300 nm), 4-thioU at position 46 forms crosslinks to pre-mRNA near the 5' splice site at nt +4, +5, +6, and +7 in the intron, whereas 4-thioU at position 51 crosslinks to the pre-mRNA at positions -2, -1, +1, +2, +3, and at the invariant G in the lariat intermediate. All crosslinks are dependent on the presence of ATP and the splicing substrate. The two crosslinks to the pre-mRNA from position 46 and 51 of U6 can also occur in prp2 heat-inactivated yeast splicing extracts blocked immediately prior to the first chemical step. Significantly, the crosslink from position 51 can undergo subsequent splicing when the mutant extract is complemented with functional Prp2 protein in a chase experiment, indicating that the crosslink reflects a functional interaction that is maintained during the first step. The crosslink to lariat intermediate appears when the mutant spliceosomes are complemented with functional Prp2 protein added exogenously. This experiment is a paradigm for future studies in which different mutant extracts are used to establish the stage in assembly at which particular RNA-RNA interactions defined by unique crosslinks occur.     

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.     

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.     

The structure of the HIV-1 RRE high affinity rev binding site at 1.6 A resolution

The crystal structure of a 28 nt RNA fragment containing the human immunodeficiency virus type 1 (HIV-1) Rev response element high affinity binding site for Rev protein has been solved at 1.6 A resolution. The overall structure of the RRE helix is greatly distorted from A-form geometry by the presence of two purine-purine base-pairs and two single nucleotide bulges. G48 and G71 form a Hoogsteen-type asymmetric base-pair with G71 adopting a syn conformation. The non-canonical regions in the unliganded Rev response element molecule narrow the major groove width with respect to standard A-RNA. The Rev response element structure observed here represents a closed form of the Rev binding site and differs from conformations of the RNA observed previously by solution NMR studies.     

Function of the human immunodeficiency virus types 1 and 2 Rev proteins is dependent on their ability to interact with a structured region present in env gene mRNA.

The interaction of the human immunodeficiency virus type 1 (HIV-1) Rev protein with a structured region in env mRNA (the Rev-responsive element [RRE]) mediates the export of structural mRNAs from the nucleus to the cytoplasm. We demonstrated that unlike HIV-1 Rev, which functions with both the HIV-1 and HIV-2 RREs, HIV-2 Rev functions only with the HIV-2 RRE. Rev-RRE binding studies suggested that the lack of nonreciprocal complementation stems from the inability of HIV-2 Rev to interact with HIV-1 RRE RNA. Maintenance of RNA secondary structure, rather than the primary nucleotide sequence, appeared to be the major determinant for interaction of both HIV-1 and HIV-2 Rev with the HIV-2 RRE. Moreover, the binding domain of the HIV-2 RRE recognized by HIV-1 Rev was dissimilar to the binding domain of the HIV-1 RRE, in terms of both secondary structure and primary nucleotide sequence. Our results support the hypothesis that function of HIV Rev proteins and possibly the functionally similar Rex proteins encoded by the human T-cell leukemia viruses (HTLVs) HTLV-I and HTLV-II is controlled by the presence of RNA secondary structure generated within the RRE RNA.     

Computational modeling suggests dimerization of equine infectious anemia virus Rev is required for RNA binding

Background

The lentiviral Rev protein mediates nuclear export of intron-containing viral RNAs that encode structural proteins or serve as the viral genome. Following translation, HIV-1 Rev localizes to the nucleus and binds its cognate sequence, termed the Rev-responsive element (RRE), in incompletely spliced viral RNA. Rev subsequently multimerizes along the viral RNA and associates with the cellular Crm1 export machinery to translocate the RNA-protein complex to the cytoplasm. Equine infectious anemia virus (EIAV) Rev is functionally homologous to HIV-1 Rev, but shares very little sequence similarity and differs in domain organization. EIAV Rev also contains a bipartite RNA binding domain comprising two short arginine-rich motifs (designated ARM-1 and ARM-2) spaced 79 residues apart in the amino acid sequence. To gain insight into the topology of the bipartite RNA binding domain, a computational approach was used to model the tertiary structure of EIAV Rev.

Results

The tertiary structure of EIAV Rev was modeled using several protein structure prediction and model quality assessment servers. Two types of structures were predicted: an elongated structure with an extended central alpha helix, and a globular structure with a central bundle of helices. Assessment of models on the basis of biophysical properties indicated they were of average quality. In almost all models, ARM-1 and ARM-2 were spatially separated by >15 Å, suggesting that they do not form a single RNA binding interface on the monomer. A highly conserved canonical coiled-coil motif was identified in the central region of EIAV Rev, suggesting that an RNA binding interface could be formed through dimerization of Rev and juxtaposition of ARM-1 and ARM-2. In support of this, purified Rev protein migrated as a dimer in Blue native gels, and mutation of a residue predicted to form a key coiled-coil contact disrupted dimerization and abrogated RNA binding. In contrast, mutation of residues outside the predicted coiled-coil interface had no effect on dimerization or RNA binding.

Conclusions

Our results suggest that EIAV Rev binding to the RRE requires dimerization via a coiled-coil motif to juxtapose two RNA binding motifs, ARM-1 and ARM-2.

     

Extending the family of UNCG-like tetraloop motifs: NMR structure of a CACG tetraloop from coxsackievirus B3

Stable RNA tetraloop motifs are found frequently in biologically active RNAs. These motifs carry out a wide variety of functions in RNA folding, in RNA-RNA and RNA-protein interactions. A great deal of knowledge about the structures and functions of tetraloop motifs has accumulated largely due to intensive theoretical, biochemical, and biophysical studies on three most frequently occurring families of tetraloop sequences, namely, the cUNCGg, the cGNRAg, and the gCUUGc sequences. Our knowledge surely is not exhaustive, and efforts are still being made to gain a better understanding. Here we report the NMR structure of a uCACGg tetraloop that occurs naturally within the cloverleaf RNA structure of the 5'-UTR of coxsackievirus B3. This tetraloop is the major determinant for interaction between the cloverleaf RNA and viral 3C protease, which is an essential part of a ribonucleoprotein complex that plays a critical role in the regulation of viral translation and replication. Our structure shows that the CACG tetraloop is closed by a wobble U.G base pair. The structure of the CACG tetraloop is stabilized by extensive base stacking and hydrogen bonding interactions strikingly similar to those previously reported for the cUUCGg tetraloop. Identification of these hallmark structural features strongly supports the existence of an extended YNCG tetraloop family. The U.G base pair closing the stem and the A residue in the loop introduce some small structural and themodynamic distinctions from the canonical cUUCGg tetraloop that may be important for recognition by the viral 3C protease.     

Analysis of the EIAV Rev-responsive element (RRE) reveals a conserved RNA motif required for high affinity Rev binding in both HIV-1 and EIAV

A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies.     

Functional Analyses Reveal Extensive RRE Plasticity in Primary HIV-1 Sequences Selected under Selective Pressure

Background

HIV-1 Rev response element (RRE) is a functional region of viral RNA lying immediately downstream to the junction of gp120 and gp41 in the env coding sequence. The RRE is essential for HIV replication and binds with the Rev protein to facilitate the export of viral mRNA from nucleus to cytoplasm. It has been suggested that changes in the predicted secondary structure of primary RRE sequences impact the function of the RREs; however, functional assays have not yet been performed. The aim of this study was to characterize the genetic, structural and functional variation in the RRE primary sequences selected in vivo by Enfuvirtide pressure.

Results

Multiple RRE variants were obtained from viruses isolated from patients who failed an Enfuvirtide-containing regimen. Different alterations were observed in the predicted RRE secondary structures, with the abrogation of the primary Rev binding site in one of the variants. In spite of this, most of the RRE variants were able to bind Rev and promote the cytoplasmic export of the viral mRNAs with equivalent efficiency in a cell-based assay. Only RRE45 and RRE40-45 showed an impaired ability to bind Rev in a gel-shift binding assay. Unexpectedly, this impairment was not reflected in functional capacity when RNA export was evaluated using a reporter assay, or during virus replication in lymphoid cells, suggesting that in vivo the RRE would be highly malleable.

Conclusions

The Rev-RRE functionality is unaffected in RRE variants selected in patients failing an ENF-containing regimen. Our data show that the current understanding of the Rev-RRE complex structure does not suffice and fails to rationally predict the function of naturally occurring RRE mutants. Therefore, this data should be taken into account in the development of antiviral agents that target the RRE-Rev complex.     

Solution structures and characterization of human immunodeficiency virus Rev responsive element IIB RNA targeting zinc finger proteins

The Rev responsive element (RRE), a part of unspliced human immunodeficiency virus (HIV) RNA, serves a crucial role in the production of infectious HIV virions. The viral protein Rev binds to RRE and facilitates transport of mRNA to the cytoplasm. Inhibition of the Rev-RRE interaction disrupts the viral life cycle. Using a phage display protocol, dual zinc finger proteins (ZNFs) were generated that bind specifically to RREIIB at the high affinity Rev binding site. These proteins were further shortened and simplified, and they still retained their RNA binding affinity. The solution structures of ZNF29 and a mutant, ZNF29G29R, have been determined by nuclear magnetic resonance (NMR) spectroscopy. Both proteins form C(2)H(2)-type zinc fingers with essentially identical structures. RNA protein interactions were evaluated quantitatively by isothermal titration calorimetry, which revealed dissociation constants (K(d)'s) in the nanomolar range. The interaction with the RNA is dependent upon the zinc finger structure; in the presence of EDTA, RNA binding is abolished. For both proteins, RNA binding is mediated by the alpha-helical portion of the zinc fingers and target the bulge region of RREIIB-TR. However, ZNF29G29R exhibits significantly stronger binding to the RNA target than ZNF29; this illustrates that the binding of the zinc finger scaffold is amenable to further improvements.