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.     

Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev

The Rev protein of HIV-1 regulates the synthesis of partially spliced forms of cytoplasmic viral mRNA by binding to a cis-acting RNA sequence, the Rev response element (RRE). We have investigated the regulation of splicing in vitro and have shown that Rev specifically inhibits splicing of pre-mRNAs containing an RRE by 3- to 4-fold. A synthetic peptide of 17 amino acids containing the RNA-binding domain of Rev is highly functional and specifically inhibits splicing by up to 30-fold. Other peptides that bind to the RRE with high affinity, but with low specificity, do not specifically inhibit splicing. Six repeated monomeric binding sites for the peptide can substitute for the RRE, indicating that regulation by Rev requires interactions with multiple sites. The peptide acts at a step in the assembly of splicing complexes, suggesting that one of the functions of the basic region of Rev is to prevent formation of a functional spliceosome.     

Mechanism of neomycin and Rev peptide binding to the Rev responsive element of HIV-1 as determined by fluorescence and NMR spectroscopy

Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short alpha-helical peptide derived from Rev (Rev 34-50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2'-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (K(D) = 20 +/- 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (K(D) = 0.24 +/- 0.040 microM), the second site inhibited Rev binding in a competitive fashion (K(D) = 1. 8 +/- 0.8 microM), and the third much weaker site (or sites) is attributed to nonspecific binding (K(D) >/= 40 microM). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and ternary complexes, and that this site is located in the lower stem region of RRE.     

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.     

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.     

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.     

Comparative analysis of Rev function in human immunodeficiency virus types 1 and 2.

The Rev proteins of the related but distinct human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) display incomplete functional reciprocity. One possible explanation for this observation is that HIV-2 Rev is unable to interact with the HIV-1 Rev-response element (RRE1). However, an analysis of the biological activity of chimeric proteins derived from HIV-1 and HIV-2 Rev reveals that this target specificity does not map to the Rev RNA binding domain but is instead primarily determined by sequences known to mediate Rev multimerization. Both HIV-1 and HIV-2 Rev are shown to bind the RRE1 in vitro with identical RNA sequence specificity. The observation that HIV-2 Rev can inhibit RRE1-dependent HIV-1 Rev function in trans indicates that the direct interaction of HIV-2 Rev with the RRE1 also occurs in vivo. These data suggest that HIV-2 Rev forms a protein-RNA complex with the RRE1 that leads to only minimal Rev activity. It is hypothesized that this low level of Rev function results from the incomplete and/or aberrant multimerization of HIV-2 Rev on this heterologous RNA target sequence.     

Binding sites for Rev and ASF/SF2 map to a 55-nucleotide purine-rich exonic element in equine infectious anemia virus RNA

The equine infectious anemia virus (EIAV) Rev protein (ERev) negatively regulates its own synthesis by inducing alternative splicing of its mRNA. This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. When Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. The interaction of ERev with its cis-acting RNA response element, the RRE, is also essential for nuclear export of intron-containing viral mRNAs that encode structural and enzymatic gene products. The primary ERev binding site and the manner in which ERev interacts with RNA or cellular proteins to exert its regulatory function have not been defined. We have performed in vitro RNA binding experiments to show that recombinant ERev binds to a 55-nucleotide, purine-rich tract proximal to the 5' splice site of exon 3. Because of its proximity to the 5' splice site and since it contains elements related to consensus exonic splicing enhancer sequences, we asked whether cellular proteins recognize the EIAV RRE. The cellular protein, ASF/SF2, a member of the serine- and arginine-rich family of splicing factors (SR proteins) bound to repeated sequences within the 55-nucleotide RRE region. Electrophoretic mobility shift and UV cross-linking experiments indicated that ERev and SR proteins bind simultaneously to the RRE. Furthermore, in vitro protein-protein interaction studies revealed an association between ERev and SR proteins. These data suggest that EIAV Rev-induced exon skipping observed in vivo may be initiated by simultaneous binding of Rev and SR proteins to the RRE that alter the subsequent assembly or catalytic activity of the spliceosomal complex.     

HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency

Expression of the structural proteins of HIV-1 requires the direct interaction of the viral Rev trans-activator with its cis-acting RNA target sequence, the Rev response element or RRE. Here, we demonstrate that this specific RNA-binding event is, as expected, mediated by the conserved arginine-rich motif of Rev. However, we also show that amino acid residues located proximal to this basic domain that are critical for in vivo Rev function are dispensable for sequence-specific binding to the RRE. Instead, these sequences are required for the multimerization of Rev on the viral RRE target sequence. The observation that Rev function requires the sequential binding of multiple Rev molecules to the RRE provides a biochemical explanation for the observed threshold effect for Rev function in vivo and suggests a molecular model for the high incidence of latent infection by HIV-1.     

HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence

Expression of human immunodeficiency virus type 1 structural proteins requires both the viral Rev trans-activator and its cis-acting RNA target sequence, the Rev response element (RRE). The RRE has been mapped to a conserved region of the HIV-1 env gene and is predicted to form a complex, highly stable RNA stem-loop structure. Site-directed mutagenesis was used to define a small subdomain of the RRE, termed stem-loop II, that is essential for biological activity. Gel retardation assays demonstrated that the Rev trans-activator is a sequence-specific RNA binding protein. The RRE stem-loop II subdomain was found to be both necessary and sufficient for the binding of Rev by the RRE. We propose that the HIV-1 Rev trans-activator belongs to a new class of sequence-specific RNA binding proteins characterized by the presence of an arginine-rich binding motif.     

RNA aptamers selected to bind human immunodeficiency virus type 1 Rev in vitro are Rev responsive in vivo.

RNA aptamers (binding sequences) that can interact tightly and specifically with the human immunodeficiency virus type 1 Rev protein have previously been selected from random sequence pools. Although the selected sequences compete with the wild-type Rev-binding element (RBE) in vitro, it was not known whether they would be able to functionally replace the RBE in vivo. Two aptamers that were different from the wild-type RBE in terms of both primary sequence and secondary structure were inserted into the full-length Rev-responsive element (RRE) in place of the RBE. The hybrid RREs were assayed for their ability to mediate Rev function in vivo using a reporter system. The aptamers were found to be functionally equivalent to the wild-type element when the assay system was saturated with Rev and better than the wild-type element when Rev was limiting. These results demonstrate that the affinity of the primary Rev-binding element rather than its particular sequence may be most responsible for conferring Rev responsiveness on viral mRNAs. Moreover, the fact that increased binding ability can lead to increased Rev responsiveness suggests that cellular factors do not directly influence the Rev:RBE interaction. Finally, since sequences distinct from the RBE are found to be Rev responsive, it may be possible for the RBE to readily mutate in response to drugs or gene therapy reagents that target the Rev:RBE interaction.     

Circular dichroism studies of the HIV-1 Rev protein and its specific RNA binding site

The circular dichroism (CD) spectrum of the Rev protein from HIV-1 indicates that Rev contains about 50% alpha helix and 25% beta sheet at 5 degrees C in potassium phosphate buffer, pH 3, and 300 mM KF. The spectrum is independent of protein concentration over a 20-fold range. At neutral pH, Rev is relatively insoluble but can be brought into solution by binding to its specific RNA binding site, the Rev-responsive element (RRE), at a Rev:RNA ratio of about 3:1. Nonspecific binding to tRNA does not solubilize Rev. As judged by difference CD spectra, the conformation of Rev when bound to the RRE at neutral pH is similar to the conformation of unbound Rev at pH 3, although changes in the RNA may also contribute to the difference spectrum. Indeed, some difference is observed near 260 nm, consistent with a conformational change of the RRE upon Rev binding. Rev alone at pH 3 shows irreversible aggregation as the temperature is raised, while Rev bound to the RRE at neutral pH shows a reversible transition with a Tm of 68 degrees C.     

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.     

Functional mapping of the human immunodeficiency virus type 1 Rev RNA binding domain: new insights into the domain structure of Rev and Rex.

Expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the direct interaction of the viral trans-activator protein Rev with its cis-acting RNA sequence (Rev-response element [RRE]). A stretch of 14 amino acid residues of the 116-amino-acid Rev protein is sufficient to impose nucleolar localization onto a heterologous protein. Our results demonstrated that these same amino acid residues confer Rev-specific RRE binding to the heterologous human T-cell leukemia virus type I Rex protein. In addition, our results indicated that amino acids distinct from the nuclear localization signal are important for Rex-specific RRE RNA binding.     

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.