Design of artificial pepetides that recognize the HIV RRE IIB RNA.

Peptide nucleic acid (PNA) conjugated peptides, derived from HIV-1 Rev, were designed and synthesized in order to construct molecules that recognize HIV RRE IIB RNA. The competitive binding analyses using fluorescent Rev peptide revealed that the PNA unit on the peptide affected the RNA binding.     

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.     

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.     

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.     

Construction of peptides with nucleobase amino acids: design and synthesis of the nucleobase-conjugated peptides derived from HIV-1 Rev and their binding properties to HIV-1 RRE RNA

In order to develop a novel molecule that recognizes a specific structure of RNA, we have attempted to design peptides having L-alpha-amino acids with a nucleobase at the side chain (nucleobase amino acid (NBA)), expecting that the function of a nucleobase which can specifically recognize a base in RNA is regulated in a peptide conformation. In this study, to demonstrate the applicability of the NBA units in the peptide to RNA recognition, we designed and synthesized a variety of NBA-conjugated peptides, derived from HIV-1 Rev. Circular dichroism study revealed that the conjugation of the Rev peptide with an NBA unit did not disturb the peptide conformation. RNA-binding affinities of the designed peptides with RRE IIB RNA were dependent on the structure of the nucleobase moieties in the peptides. The peptide having the cytosine NBA at the position of the Asn40 site in the Rev showed a higher binding ability for RRE IIB RNA, despite the diminishing the Asn40 function. Furthermore, the peptide having the guanine NBA at the position of the Arg44 site, which is the most important residue for the RNA binding in the Rev, bound to RRE IIB RNA in an ability similar to Rev34-50 with native sequence. These results demonstrate that an appropriate NBA unit in the peptide plays an important role in the RNA binding with a specific contact such as hydrogen bonding, and the interaction between the nucleobase in the peptide and the base in the RNA can enhance the RNA-binding affinity and specificity.     

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.     

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.     

Cooperative dimerization of a stably folded protein directed by a flexible RNA in the assembly of the HIV Rev dimer–RRE stem II complex

The binding of the HIV‐1 Rev protein as an oligomer to a viral RNA element, the Rev‐response element (RRE), mediates nuclear export of genomic RNA. Assembly of the Rev–RRE ribonucleoprotein (RNP) complex is nucleated by the binding of the first Rev molecule to stem IIB of the RRE. This is followed by stepwise addition of a total of ~six Rev molecules along the RRE through a combination of RNA–protein and protein–protein interactions. RRE stem II, which forms a three‐way junction consisting of stems IIA, IIB and IIC, has been shown to bind to two Rev molecules in a cooperative manner, with the second Rev molecule binding to the junction region of stem II. The results of base substitutions at the stem II junction, and characterization of stem II junction variants selected from a randomized library showed that an “open” flexible structure is preferred for binding of the second Rev molecule, and that binding of the second Rev molecule to the junction region is not sequence‐specific. Alanine substitutions of a number of Rev amino acid residues implicated to be important for Rev folding in previous structural studies were found to result in a dramatic decrease in the binding of the second Rev molecule. These results support the model that proper folding of Rev is critical in ensuring that the flexible RRE is able to correctly position Rev molecules for specific RNP assembly, and suggests that targeting Rev folding may be effective in the inhibition of Rev function. Copyright © 2015 John Wiley & Sons, Ltd.     

A solution to limited genomic capacity: using adaptable binding surfaces to assemble the functional HIV Rev oligomer on RNA

Many ribonucleoprotein (RNP) complexes assemble into large, organized structures in which protein subunits are positioned by interactions with RNA and other proteins. Here we demonstrate that HIV Rev, constrained in size by a limited viral genome, also forms an organized RNP by assembling a homo-oligomer on the Rev response element (RRE) RNA. Rev subunits bind cooperatively to discrete RNA sites using an oligomerization domain and an adaptable protein-RNA interface, forming a complex with 500-fold higher affinity than the tightest single interaction. High-affinity binding correlates strongly with RNA export activity. Rev utilizes different surfaces of its alpha-helical RNA-binding domain to recognize several low-affinity binding sites, including the well-characterized stem IIB site and an additional site in stem IA. We propose that adaptable RNA-binding surfaces allow the Rev oligomer to assemble economically into a discrete, stable RNP and provide a mechanistic role for Rev oligomerization during the HIV life cycle.     

Specific RNA binding by a single C2H2 zinc finger

Zinc finger proteins with high affinity for human immunodeficiency virus Rev responsive element stem loop IIB (RRE-IIB) were previously isolated from a phage display zinc finger library. Zinc fingers from one of these proteins, RR1, were expressed individually and assayed for RRE-IIB affinity. The C-terminal zinc finger retained much of the binding affinity of the two-finger parent and was disrupted by mutations predicted to narrow the RRE-IIB major groove and which disrupt Rev binding. In contrast, the N-terminal zinc finger has a calculated affinity at least 1000-fold lower. Despite the high affinity and specificity of RR1 for RRE-IIB, binding affinity for a 234-nucleotide human immunodeficiency virus Rev responsive element (RRE234) was significantly lower. Therefore, zinc finger proteins that bind specifically to RRE234 were constructed using an in vitro selection and recombination approach. These zinc fingers bound RRE234 with subnanomolar dissociation constants and bound the isolated RRE-IIB stem loop with an affinity 2 orders of magnitude lower but similar to the affinity of an arginine-rich peptide derived from Rev. These data show that single C2H2 zinc fingers can bind RNA specifically and suggest that their binding to stem loop IIB is similar to that of Rev peptide. However, binding to RRE234 is either different from stem loop IIB binding or the tertiary structure of stem loop IIB is changed within the Rev responsive element.     

Amino acid requirement for the high affinity binding of a selected arginine-rich peptide with the HIV Rev-response element RNA.

The arginine-rich motif is a class of short arginine-rich peptides that bind to specific RNA structures that has been found to be a versatile framework for the design and selection of RNA-binding peptides. We previously identified novel peptides that bind to the Rev-response element (RRE) RNA of the HIV from an arginine-rich polypeptide library (ARPL) consisting of a polyarginine (15 mer) randomized at the N-terminal 10 positions. The selected peptides bound more strongly to the RRE than the natural binding partner, Rev, and contained glutamine residues that were assumed to be important for recognition of the G-A base pair. In addition, the peptides were predicted to bind to the RRE in an alpha-helical conformation. In this study, in order to understand the mechanism of the interaction between the RRE and the putative alpha-helical glutamine-containing peptides, the amino acid requirements for high affinity binding were analyzed by a combinatorial approach using a bacterial system for detecting RNA-peptide interactions. A consensus peptide, the DLA peptide, was elucidated, which consists of a single glutamine residue within a polyarginine context with the glutamine residue flanked at specific positions by three nonarginine residues, two of which appear to be important for alpha-helix stabilization. In addition, the DLA peptide was found to bind extremely tightly to the RRE with an affinity 50-fold higher than that of the Rev peptide as determined by a gel shift assay. A working model for the interaction of the DLA peptide to the RRE is proposed, which should aid in the development of peptide-based drugs that inhibit HIV replication, as well as in our understanding of polypeptide-RNA interactions. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibition of nuclear import mediated by the Rev-arginine rich motif by RNA molecules

The HIV-1 Rev protein plays a pivotal role in viral replication, and therefore, inhibition of its function should block the progression of the virus-induced immune deficiency syndrome (AIDS). Here, RNA molecules have been shown to inhibit import of the HIV-1 Rev protein into nuclei of permeabilized cells. Nuclear uptake of biotinylated recombinant His-tagged Rev-GFP was assessed in nuclear extracts from digitonin-permeabilized cells by binding to either importin beta-receptors or nickel molecules immobilized on a microtiter plate. Using this method together with fluorescence microscopy, we determined that nuclear import of Rev is inhibited by the addition of a reticulocyte lysate which routinely is used as a source of nuclear import receptors. This inhibition was released by treatment with the RNase enzyme. Also t-RNA molecules and the oligoribonucleotide RRE IIB, namely, the second stem structure of the Rev responsive element (RRE) of the viral RNA, inhibit Rev nuclear import. Similar results were obtained when BSA molecules with covalently attached Rev-arginine rich motif (ARM) peptides were used as a nuclear transport substrate, indicating that the nuclear import inhibition of the Rev protein is due to the presence of the ARM domain. Binding experiments revealed that the RNA molecules inhibit the interaction between the ARM region and importin beta, implying that the RNA prevents the formation of the import complex. The implication of our results for the regulation of the nuclear import of Rev as well as for the use of RNA molecules as antiviral drugs is discussed.     

Specific binding of a basic peptide from HIV-1 Rev.

Human immunodeficiency virus type I (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev activity is mediated through specific binding to a cis-acting Rev responsive element (RRE) located within the env region of HIV-1. A monomer Rev binding site corresponding to 37 nucleotides of the RRE (IIB RNA) was studied by RNA footprinting, modification interference experiments and mutational analysis. Surprisingly, a 17 amino acid peptide, corresponding to the basic domain of Rev, binds specifically to this site at essentially identical nucleotides and probably induces additional base pairing. The Rev protein and related peptide interact primarily with two sets of nucleotides located at the junction of single and double stranded regions, and at an additional site located within a helix. This suggests that the domains of proteins responsible for specific RNA binding can be remarkably small and that the interaction between RNA and protein can probably induce structure in both constituents.     

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.     

Chimeric RNase H-competent oligonucleotides directed to the HIV-1 Rev response element

Chimeric oligo-2'-O-methylribonucleotides containing centrally located patches of contiguous 2'-deoxyribonucleotides and terminating in a nuclease resistant 3'-methylphosphonate internucleotide linkage were prepared. The oligonucleotides were targeted to the 3'-side of HIV Rev response element (RRE) stem-loop IIB RNA, which is adjacent to the high affinity Rev protein binding site and is critical to virus function. Thermal denaturation experiments showed that chimeric oligonucleotides form very stable duplexes with a complementary single-stranded RNA, and gel electrophoretic mobility shift assays (EMSA) showed that they bind with high affinity and specificity to RRE stem-loop II RNA (K(D) approximately 200 nM). The chimeric oligonucleotides promote RNase H-mediated hydrolysis of RRE stem-loop II RNA and have half-lives exceeding 24h when incubated in cell culture medium containing 10% fetal calf serum. One of the chimeric oligonucleotides inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) approximately 60% at a concentration of 300 nM in HEK 293T cells co-transfected with p-RRE/CAT and p-Rev mammalian expression vectors.     

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.     

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.     

The HIV-2 Rev-response element: determining secondary structure and defining folding intermediates

Interaction between the viral protein Rev and the RNA motifs known as Rev response elements (RREs) is required for transport of unspliced and partially spliced human immunodeficiency virus (HIV)-1 and HIV-2 RNAs from the nucleus to the cytoplasm during the later stages of virus replication. A more detailed understanding of these nucleoprotein complexes and the host factors with which they interact should accelerate the development of new antiviral drugs targeting cis-acting RNA regulatory signals. In this communication, the secondary structures of the HIV-2 RRE and two RNA folding precursors have been identified using the SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing methodology together with a novel mathematical approach for determining the secondary structures of RNA conformers present in a mixture. A complementary chemical probing technique was also used to support these secondary structure models, to confirm that the RRE2 RNA undergoes a folding transition and to obtain information about the relative positioning of RRE2 substructures in three dimensions. Our analysis collectively suggests that the HIV-2 RRE undergoes two conformational transitions before assuming the energetically most favorable conformer. The 3D models for the HIV-2 RRE and folding intermediates are also presented, wherein the Rev-binding stem–loops (IIB and I) are located coaxially in the former, which is in agreement with previous models for HIV-1 Rev-RRE binding.