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.     

A highly conserved RNA folding region coincident with the Rev response element of primate immunodeficiency viruses.

A series of unusual folding regions (UFR) immediately 3' to the cleavage site of the outer membrane protein (OMP) and transmembrane protein (TMP) were detected in the envelope gene RNA of the human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV) by an extensive Monte Carlo simulation. These RNA secondary structures were predicted to be both highly stable and statistically significant. In the calculation, twenty-five different sequence isolates of HIV-1, three isolates of HIV-2 and eight sequences of SIV were included. Although significant sequence divergence occurs in the env coding regions of these viruses, a distinct UFR of 234-nt is consistently located ten nucleotides 3' to the cleavage site of the OMP/TMP in HIV-1, and a 216-nt UFR occurs forty-six and forty-nine nucleotides downstream from the OMP/TMP cleavage site of HIV-2 and SIV, respectively. Compensatory base changes in the helical stem regions of these conserved RNA secondary structures are identified. These results support the hypothesis that these special RNA folding regions are functionally important and suggest that the role of this sequence as the Rev response element (RRE) is mediated by secondary structure as well as primary RNA sequence.     

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.     

The HIV-1 Rev response element (RRE) adopts alternative conformations that promote different rates of virus replication

The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, we have now determined that the wt NL4-3 RRE exists as a mixture of both structures. To assess functional differences between these RRE ‘conformers’, we created conformationally locked mutants by site-directed mutagenesis. Using subgenomic reporters, as well as HIV replication assays, we demonstrate that the five stem-loop form of the RRE promotes greater functional Rev/RRE activity compared to the four stem-loop counterpart.     

Sam68 is absolutely required for Rev function and HIV-1 production

Sam68 functionally complements for, as well as synergizes with, HIV-1 Rev in Rev response element (RRE)-mediated gene expression and virus production. Furthermore, C-terminal deletion/point mutants of Sam68 (Sam68ΔC/Sam68-P21) exert a transdominant negative phenotype for Rev function and HIV-1 production. However, the relevance of Sam68 in Rev/RRE function is not well defined. To gain more insight into the mechanism of Sam68 in Rev function, we used an RNAi (RNA interference) strategy to create stable Sam68 knockdown HeLa (SSKH) cells. In SSKH cells, Rev failed to activate both RRE-mediated reporter gene [chloramphenicol acetyltransferase (CAT) and/or gag] expressions. Importantly, reduction of Sam68 expression led to a dramatic inhibition of HIV-1 production. Inhibition of the reporter gene expression and HIV production correlated with the failure to export RRE-containing CAT mRNA and unspliced viral mRNAs to the cytoplasm, confirming that SSKH cells are defective for Rev-mediated RNA export. Taken together, these results suggest that Sam68 is involved in Rev-mediated RNA export and is absolutely required for HIV production.     

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.     

Functional Analysis of the Human Immunodeficiency Virus Type 1 Rev Protein Oligomerization Interface

The expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the action of the viral trans-regulatory protein Rev. Rev is a nuclear shuttle protein that directly binds to its cis-acting Rev response element (RRE) RNA target sequence. Subsequent oligomerization of Rev monomers on the RRE and interaction of Rev with a cellular cofactor(s) result in the cytoplasmic accumulation of RRE-containing viral mRNAs. Moreover, Rev by itself is exported from the nucleus to the cytoplasm. Although it has been demonstrated that Rev multimerization is critically required for Rev activity and hence for HIV-1 replication, the number of Rev monomers required to form a trans-activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev trans-activator protein. We identify the amino acid residues which are part of the proposed single hydrophobic surface patch in the Rev amino terminus that mediates intermolecular interactions. Furthermore, we show that the expression of a multimerization-deficient Rev mutant blocks HIV-1 replication in a trans-dominant (dominant-negative) fashion.     

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.     

Multiple copies of the Mason-Pfizer monkey virus constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent manner

Retroviral gene expression requires nuclear export and translation of incompletely spliced RNA. In the case of human immunodeficiency virus (HIV), this is facilitated by the viral Rev protein binding to its cognate RNA response element (RRE), while other retroviruses contain constitutive transport elements (CTE) binding to cellular factors. These CTE can substitute for the HIV-1 Rev/RRE system, albeit with reduced efficiency. Here, we show that multimeric copies of the CTE restore HIV-1 protein expression to levels comparable to or higher than Rev/RRE in various cell lines from different species. We suggest that multimerization of export factors is important for CTE function, as reported for Rev. CTE function was not affected when the element was displaced from its natural position close to the poly(A) signal, while insertion of an intron into the 3′-untranslated region (3′-UTR) severely reduced CTE activity. In this case, cytoplasmic RNA degradation was observed, which may be mediated by nonsense-mediated RNA decay. In contrast, Rev-dependent gene expression was insensitive to an intron in the 3′-UTR. Finally, we show that the putative CTE-binding protein RNA helicase A is not specifically translocated into the cytoplasm upon overexpression of CTE-containing RNA.     

Cross-activation of the Rex proteins of HTLV-I and BLV and of the Rev protein of HIV-1 and nonreciprocal interactions with their RNA responsive elements

The Rex regulatory proteins of human T-cell leukemia virus type I (HTLV-I) and bovine leukemia virus (BLV), and the Rev protein of human immunodeficiency virus type 1 (HIV-1), promote the cytoplasmic accumulation and translation of viral messenger mRNAs encoding structural proteins. Rev and Rex act through cis-acting elements on the viral RNA; these elements are named Rev- and Rex-responsive elements, or RRE and RXRE, respectively. We show that the Rex proteins of HTLV-I and BLV are interchangeable, but only the Rex protein of HTLV-I can substitute for Rev of HIV-1. Rex of HTLV-I and Rev of HIV-1 appear to act on RRE by similar mechanisms. Rev of HIV-1 does not act on the RXRE of HTLV-I or BLV. The nonreciprocal action of Rev and Rex suggests that these factors interact directly with the cis-acting RNA elements of the two viruses.     

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.     

Rev proteins of human and simian immunodeficiency virus enhance RNA encapsidation

The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Using Rev-independent gag-pol expression plasmids of HIV-1 and simian immunodeficiency virus and lentiviral vector constructs, we have observed that HIV-1 and simian immunodeficiency virus Rev enhanced RNA encapsidation 20- to 70-fold, correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. In addition to specific interactions of nucleocapsid with the packaging signal at the 5' end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral 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.     

Dynamics of RNA-protein interactions in the HIV-1 Rev-RRE complex visualized by 6-thioguanosine-mediated photocrosslinking.

Expression of the structural proteins of human immunodeficiency virus type 1 (HIV-1) requires the direct interaction of multiple copies of the viral protein Rev with its target RNA, the Rev response element (RRE). RRE is a complex 351-nt RNA that is highly structured and located within the viral env gene. During initial Rev-RRE recognition, Rev binds with high affinity to a bubble structure located within the RRE RNA stem-loop II. We have used a site-specific photocrosslinking method based on 6-thioguanosine (6-thioG) photochemistry to probe the conformation of the high-affinity binding site of RRE RNA and its interactions with Rev protein under physiological conditions. A minimal duplex RNA containing the bubble region of RRE and 12 flanking base pairs was synthesized chemically. Two different RRE constructs with a single photoactive nucleoside (6-thio-dG or 6-thioG) at position 47 or 48 were synthesized. Upon UV irradiation, 6-thioG at both positions formed interstrand covalent crosslinks in RRE RNA. Mapping of crosslink sites by RNA sequencing revealed that 6-thioG at position 47 or 48 crosslinked to A73. In the presence of Rev, both RNA-RNA and RNA-protein crosslinks were observed, however, the RNA-RNA crosslink site was unchanged. Our results provide direct evidence that, during RNA-protein recognition, Rev is in close proximity to O6 of G47 and G48 in the major groove of RRE RNA. Our results also show that the bubble region of RRE RNA has a biologically relevant structure where G47 and G48 are in close proximity to A73 and this RNA structure is not changed significantly upon Rev binding. We propose that Rev protein recognizes and binds to specific structural elements of RRE RNA containing non-Watson-Crick base pairs and such structures could be a determinant for recognition by other RNA-binding proteins. Our site-specific crosslinking methods provide a general approach to capture dynamic states of biologically relevant RNA structures that are otherwise missed by NMR and X-ray crystallographic studies.     

Hidden messages in the nef gene of human immunodeficiency virus type 1 suggest a novel RNA secondary structure

The coexistence of multiple codes in the genome of human immunodeficiency virus type 1 (HIV-1) was analyzed. We explored factors constraining the variability of the virus genome primarily in relation to conserved RNA secondary structures overlapping coding sequences, and used a simple combination of algorithms for RNA secondary structure prediction based on the nearest-neighbor thermodynamic rules and a statistical approach. In our previous study, we applied this combination to a non- redundant data set of env nucleotide sequences, confirmed the conservative secondary structure of the rev-responsive element (RRE) and found a new RNA structure in the first conserved (C1) region of the env gene. In this study, we analyzed the variability of putative RNA secondary structures inside the nef gene of HIV-1 by applying these algorithms to a non-redundant data set of 104 nef sequences retrieved from the Los Alamos HIV database, and predicted the existence of a novel functional RNA secondary structure in the β3/β4 regions of nef. The predicted RNA fold in the β3/β4 region of nef appears in two forms with different loop sizes. The loop of the first fold consists of seven nucleotides (positions 494–500), with consensus UCAAGCU appearing in 79% of sequences. The other has a five-base loop (positions 495–499) with consensus CAAGC. The difference in size between these two loops may reflect the difference between respective counterparts in the hairpin recognition. This may also have an adaptive biological significance.     

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.     

Trafficking through the Rev/RRE pathway is essential for efficient inhibition of human immunodeficiency virus type 1 by an antisense RNA derived from the envelope gene

A human immunodeficiency virus type 1 (HIV-1)-based vector expressing an antisense RNA directed against HIV-1 is currently in clinical trials. This vector has shown a remarkable ability to inhibit HIV-1 replication, in spite of the fact that therapeutic use of unmodified antisense RNAs has generally been disappointing. To further analyze the basis for this, we examined the effects of different plasmid-based HIV-1 long-terminal-repeat-driven constructs expressing antisense RNA to the same target region in HIV-1 but containing different export elements. Two of these vectors were designed to express antisense RNA containing either a Rev response element (RRE) or a Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE). In the third vector, no specific transport element was provided. Efficient inhibition of HIV-1 virus production was obtained with the RRE-driven antisense RNA. This construct also efficiently inhibited p24 production from a pNL4-3 provirus that used the MPMV CTE for RNA export. In contrast, little inhibition was observed with the constructs lacking an RRE. Furthermore, when the RRE-driven antisense RNA was redirected to the Tap/Nxf1 pathway, utilized by the MPMV CTE, through the expression of a RevM10-Tap fusion protein, the efficiency of antisense inhibition was greatly reduced. These results indicate that efficient inhibition requires trafficking of the antisense RNA through the Rev/RRE pathway. Mechanistic studies indicated that the Rev/RRE-mediated inhibition did not involve either nuclear retention or degradation of target mRNA, since target RNA was found to export and associate normally with polyribosomes. However, protein levels were significantly reduced. Taken together, our results suggest a new mechanism for antisense inhibition of HIV mediated by Rev/RRE.