Structure of HIV-1 RRE stem-loop II identifies two conformational states of the high-affinity Rev binding site.

During HIV infection, specific RNA-protein interaction between the Rev response element (RRE) and viral Rev protein is required for nuclear export of intron-containing viral mRNA transcripts. Rev initially binds the high-affinity site in stem-loop II, which promotes oligomerization of additional Rev proteins on RRE. Here, we present the crystal structure of RRE stem-loop II in distinct closed and open conformations. The high-affinity Rev-binding site is located within the three-way junction rather than the predicted stem IIB. The closed and open conformers differ in their non-canonical interactions within the three-way junction, and only the open conformation has the widened major groove conducive to initial Rev interaction. Rev binding assays show that RRE stem-loop II has high- and low-affinity binding sites, each of which binds a Rev dimer. We propose a binding model, wherein Rev-binding sites on RRE are sequentially created through structural rearrangements induced by Rev-RRE interactions.

An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection.

Tat/Rev Induced Limiting Dilution Assay (TILDA) is instrumental in estimating the size of latent reservoirs of HIV-1. Here, we report an optimized TILDA containing a broader detection range compared to the reported methods and high sensitivity. Giving priority to sequence conservation, we positioned the two forward primers and the probe in exon-1 of HIV-1. The reverse primers are positioned in highly conserved regions of exon-7. The optimized TILDA detected eight molecular clones belonging to five major genetic subtypes of HIV-1 with a comparable detection sensitivity. Using the optimized assay, we show that only a minor proportion of CD4+ T cells of primary clinical samples can spontaneously generate multiply spliced viral transcripts. A significantly larger proportion of the cells produced viral transcripts following activation. The optimized TILDA is suitable to characterize HIV-1 latent reservoirs and the therapeutic strategies intended to target the reservoir size.

Native mass spectrometry reveals the initial binding events of HIV-1 rev to RRE stem II RNA.

Nuclear export complexes composed of rev response element (RRE) ribonucleic acid (RNA) and multiple molecules of rev protein are promising targets for the development of therapeutic strategies against human immunodeficiency virus type 1 (HIV-1), but their assembly remains poorly understood. Using native mass spectrometry, we show here that rev initially binds to the upper stem of RRE IIB, from where it is relayed to binding sites that allow for rev dimerization. The newly discovered binding region implies initial rev recognition by nucleotides that are not part of the internal loop of RRE stem IIB RNA, which was previously identified as the preferred binding region. Our study highlights the unique capability of native mass spectrometry to separately study the binding interfaces of RNA/protein complexes of different stoichiometry, and provides a detailed understanding of the mechanism of RRE/rev association with implications for the rational design of potential drugs against HIV-1 infection.

Nucleic acid recognition and antiviral activity of 1,4-substituted terphenyl compounds mimicking all faces of the HIV-1 Rev protein positively-charged α-helix.

Small synthetic molecules mimicking the three-dimensional structure of α-helices may find applications as inhibitors of therapeutically relevant protein-protein and protein-nucleic acid interactions. However, the design and use of multi-facial helix mimetics remains in its infancy. Here we describe the synthesis and application of novel bilaterally substituted p-terphenyl compounds containing positively-charged aminoalkyl groups in relative 1,4 positions across the aromatic scaffold. These compounds were specifically designed to mimic all faces of the arginine-rich α-helix of the HIV-1 protein Rev, which forms deeply embedded RNA complexes and plays key roles in the virus replication cycle. Two of these molecules recognized the Rev site in the viral RNA and inhibited the formation of the RRE-Rev ribonucleoprotein complex, a currently unexploited target in HIV chemotherapy. Cellular assays revealed that the most active compounds blocked HIV-1 replication with little toxicity, and likely exerted this effect through a multi-target mechanism involving inhibition of viral LTR promoter-dependent transcription and Rev function. Further development of this scaffold may open new avenues for targeting nucleic acids and may complement current HIV therapies, none of which involve inhibitors interfering with the gene regulation processes of the virus.

Structural Fluidity of the Human Immunodeficiency Virus Rev Response Element.

Nucleocytoplasmic transport of unspliced and partially spliced human immunodeficiency virus (HIV) RNA is mediated in part by the Rev response element (RRE), a ~350 nt cis-acting element located in the envelope coding region of the viral genome. Understanding the interaction of the RRE with the viral Rev protein, cellular co-factors, and its therapeutic potential has been the subject of almost three decades of structural studies, throughout which a recurring discussion theme has been RRE topology, i.e., whether it comprises 4 or 5 stem-loops (SLs) and whether this has biological significance. Moreover, while in vitro mutagenesis allows the construction of 4 SL and 5 SL RRE conformers and testing of their roles in cell culture, it has not been immediately clear if such findings can be translated to a clinical setting. Herein, we review several articles demonstrating remarkable flexibility of the HIV-1 and HIV-2 RREs following initial observations that HIV-1 resistance to trans-dominant Rev therapy was founded in structural rearrangement of its RRE. These observations can be extended not only to cell culture studies demonstrating a growth advantage for the 5 SL RRE conformer but also to evolution in RRE topology in patient isolates. Finally, RRE conformational flexibility provides a target for therapeutic intervention, and we describe high throughput screening approaches to exploit this property.

m6A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB.

N6-methyladenosine (m6A) is a ubiquitous RNA post-transcriptional modification found in coding as well as non-coding RNAs. m6A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m6A sites have been reported in the HIV-1 Rev response element (RRE) stem IIB, one of which was shown to enhance binding to the viral protein Rev and viral RNA export. However, because these m6A sites have not been observed in other studies mapping m6A in HIV-1 RNA, their significance remains to be firmly established. Here, using optical melting experiments, NMR spectroscopy, and in vitro binding assays, we show that m6A minimally impacts the stability, structure, and dynamics of RRE stem IIB as well as its binding affinity to the Rev arginine-rich-motif (ARM) in vitro. Our results indicate that if present in stem IIB, m6A is unlikely to substantially alter the conformational properties of the RNA. Our results add to a growing view that the impact of m6A on RNA depends on sequence context and Mg2+.

Highly Mutable Linker Regions Regulate HIV-1 Rev Function and Stability.

HIV-1 Rev is an essential viral regulatory protein that facilitates the nuclear export of intron-containing viral mRNAs. It is organized into structured, functionally well-characterized motifs joined by less understood linker regions. Our recent competitive deep mutational scanning study confirmed many known constraints in Rev's established motifs, but also identified positions of mutational plasticity, most notably in surrounding linker regions. Here, we probe the mutational limits of these linkers by testing the activities of multiple truncation and mass substitution mutations. We find that these regions possess previously unknown structural, functional or regulatory roles, not apparent from systematic point mutational approaches. Specifically, the N- and C-termini of Rev contribute to protein stability; mutations in a turn that connects the two main helices of Rev have different effects in different contexts; and a linker region which connects the second helix of Rev to its nuclear export sequence has structural requirements for function. Thus, Rev function extends beyond its characterized motifs, and is tuned by determinants within seemingly plastic portions of its sequence. Additionally, Rev's ability to tolerate many of these massive truncations and substitutions illustrates the overall mutational and functional robustness inherent in this viral protein.

Computational Studies of Intrinsically Disordered Proteins.

Frequently elusive to experimental characterizations, intrinsically disordered proteins (IDPs) can be probed using molecular dynamics to provide detailed insight into their complex structure, dynamics, and function. However, previous computational studies were often found to disagree with experiment due to either force field biases or insufficient sampling. In this study, nine unstructured short peptides and the HIV-1 Rev protein were simulated and extended to microseconds to assess these limitations in IDP simulations. In short peptide simulations, a tested IDP-specific force field ff14IDPSFF outperforms its generic counterpart ff14SB as agreement of simulated NMR observables with experiment improves, though its advantages are not clear-cut in apo Rev simulations. It is worth noting that sampling is probably still not sufficient in the ff14SB simulations of apo Rev even if 10 ms have been collected. This indicates that enhanced sampling techniques would greatly benefit IDP simulations. Finally, detailed structural analyses of apo Rev conformations demonstrate different secondary structural preferences between ff14SB (helical) and ff14IDPSFF (random coil). A natural next step is to ask a more quantitative question: whether ff14SB is too ordered or ff14IDPSFF is too disordered in simulations of more complex IDPs such as Rev. This requires further quantitative analyses both experimentally and computationally.

Highly efficient cellular uptake of a cell-penetrating peptide (CPP) derived from the capsid protein of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is one of the smallest, nonenveloped, single-stranded DNA viruses. The PCV2 capsid protein (Cap) is the sole viral structural protein and main antigenic determinant. Previous sequence analysis has revealed that the N terminus of the PCV2 Cap contains a nuclear localization signal (NLS) enriched in positively charged residues. Here, we report that PCV2's NLS can function as a cell-penetrating peptide (CPP). We observed that this NLS can carry macromolecules, e.g. enhanced GFP (EGFP), into cells when they are fused to the NLS, indicating that it can function as a CPP, similar to the classical CPP derived from HIV type 1 transactivator of transcription protein (HIV TAT). We also found that the first 17 residues of the NLS (NLS-A) have a key role in cellular uptake. In addition to entering cells via multiple endocytic processes, NLS-A was also rapidly internalized via direct translocation enabled by increased membrane permeability and was evenly distributed throughout cells when its concentration in cell cultures was ≥10 µm Of note, cellular NLS-A uptake was ∼10 times more efficient than that of HIV TAT. We inferred that the externalized NLS of the PCV2 Cap may accumulate to a high concentration (≥10 µm) at a local membrane area, increasing membrane permeability to facilitate viral entry into the cell to release its genome into a viral DNA reproduction center. We conclude that NLS-A has potential as a versatile vehicle for shuttling foreign molecules into cells, including pharmaceuticals for therapeutic interventions.

Structure of an RNA Aptamer that Can Inhibit HIV-1 by Blocking Rev-Cognate RNA (RRE) Binding and Rev-Rev Association.

HIV-1 Rev protein mediates nuclear export of unspliced and partially spliced viral RNAs for production of viral genomes and structural proteins. Rev assembles on a 351-nt Rev response element (RRE) within viral transcripts and recruits host export machinery. Small (<40-nt) RNA aptamers that compete with the RRE for Rev binding inhibit HIV-1 viral replication. We determined the X-ray crystal structure of a potential anti-HIV-1 aptamer that binds Rev with high affinity (Kd = 5.9 nM). The aptamer is structurally similar to the RRE high-affinity site but forms additional contacts with Rev unique to its sequence. Exposed bases of the aptamer interleave with the guanidinium groups of two arginines of Rev, forming stacking interactions and hydrogen bonds. The aptamer also obstructs an oligomerization interface of Rev, blocking Rev self-assembly. We propose that this aptamer can inhibit HIV-1 replication by interfering with Rev-RRE, Rev-Rev, and possibly Rev-host protein interactions.

Single-molecule fluorescence imaging: Generating insights into molecular interactions in virology.

Single-molecule fluorescence methods remain a challenging yet information-rich set of techniques that allow one to probe the dynamics, stoichiometry and conformation of biomolecules one molecule at a time. Viruses are small (nanometers) in size, can achieve cellular infections with a small number of virions and their lifecycle is inherently heterogeneous with a large number of structural and functional intermediates. Single-molecule measurements that reveal the complete distribution of properties rather than the average can hence reveal new insights into virus infections and biology that are inaccessible otherwise. This article highlights some of the methods and recent applications of single-molecule fluorescence in the field of virology. Here, we have focused on new findings in virus-cell interaction, virus cell entry and transport, viral membrane fusion, genome release, replication, translation, assembly, genome packaging, egress and interaction with host immune proteins that underline the advantage of single-molecule approach to the question at hand. Finally, we discuss the challenges, outlook and potential areas for improvement and future use of single-molecule fluorescence that could further aid our understanding of viruses.

Protein Regulation by Intrinsically Disordered Regions: A Role for Subdomains in the IDR of the HIV-1 Rev Protein.

Intrinsically disordered regions (IDRs) in proteins are highly abundant, but they are still commonly viewed as long stretches of polar, solvent-accessible residues. Here we show that the disordered C-terminal domain (CTD) of HIV-1 Rev has two subregions that carry out two distinct complementary roles of regulating protein oligomerization and contributing to stability. We propose that this takes place through a delicate balance between charged and hydrophobic residues within the IDR. This means that mutations in this region, as well as the known mutations in the structured region of the protein, can affect protein function. We suggest that IDRs in proteins should be divided into subdomains similarly to structured regions, rather than being viewed as long flexible stretches.

A DEAD-Box Helicase Mediates an RNA Structural Transition in the HIV-1 Rev Response Element.

Nuclear export of partially spliced or unspliced HIV-1 RNA transcripts requires binding of the viral protein regulator of expression of virion (Rev) to the Rev response element (RRE) and subsequent oligomerization in a cooperative manner. Cellular DEAD-box helicase DEAD-box protein 1 (DDX1) plays a role in HIV replication, interacting with and affecting Rev-containing HIV transcripts in vivo, interacting directly with the RRE and Rev in vitro, and promoting Rev oligomerization in vitro. Binding of DDX1 results in enhancement of Rev oligomerization on the RRE that is correlated with an RNA structural change within the RRE that persists even after dissociation of DDX1. Furthermore, this structural transition is likely located within the three-way junction of stem II of the RRE that is responsible for initial Rev binding. This discovery of the stem II structural transition leads to a model wherein DDX1 can act as an RNA chaperone, folding stem IIB into a proper Rev binding conformation.

Binding and thermodynamics of REV peptide-ctDNA interaction.

The thermodynamics of DNA-ligand binding is important as it provides useful information to understand the details of binding processes. HIV-1 REV response element (RRE) located in the env coding region of the viral genome is reported to be well conserved across different HIV-1 isolates. In this study, the binding characteristics of Calf thymus DNA (ctDNA) and REV peptide from HIV-1 were investigated using spectroscopic (UV-visible, fluorescence, and circular dichroism (CD)) and isothermal titration calorimetric (ITC) techniques. Thermal stability and ligand binding properties of the ctDNA revealed that native ctDNA had a Tm of 75.5 °C, whereas the ctDNA-REV peptide complex exhibited an incremental shift in the Tm by 8 °C, indicating thermal stability of the complex. CD data indicated increased ellipticity due to large conformational changes in ctDNA molecule upon binding with REV peptide and two binding stoichiometric modes are apparent. The ctDNA experienced condensation due to large conformational changes in the presence of REV peptide and positive Bâ†’Ψ transition was observed at higher molar charge ratios. Fluorescence studies performed at several ligand concentrations revealed a gradual decrease in the fluorescence intensity of EtBr-bound ctDNA in response to increasing ligand concentrations. The fluorescence data further confirmed two stoichiometric modes of binding for ctDNA-REV peptide complex as previously observed with CD studies. The binding enthalpies were determined using ITC in the temperature range of 293 K-308 K. The ITC binding isotherm was exothermic at all temperatures examined, with low ΔH values indicating that the ctDNA-REV peptide interaction is driven largely by entropy. The heat capacity change (ΔCp ) was insignificant, an unusual finding in the area of DNA-peptide interaction studies. The variation in the values obtained for ΔH, ΔS, and ΔG with temperature further suggests that ctDNA-REV peptide interaction is entropically driven. ITC based analysis of salt dependence of binding constant gave a charge value (Z) = +4.01, as determined for the δlnK/δln[Na+ ] parameter, suggesting the participation of only 3-4 Arg out of 11 Arg charge from REV peptide. The stoichiometry observed for the complex was three molar charge of REV peptide binding per molar charge of ctDNA. ITC based analysis further confirmed that the binding between ctDNA and REV peptide is governed by electrostatic interaction. Molecular interactions including H-bonding, van der Waals forces, and solvent molecules rearrangement, underlie the binding of REV peptide to ctDNA.

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export.

HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. IMPORTANCE: HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.

The Structure of HIV-1 Rev Filaments Suggests a Bilateral Model for Rev-RRE Assembly.

HIV-1 Rev protein mediates the nuclear export of viral RNA genomes. To do so, Rev oligomerizes cooperatively onto an RNA motif, the Rev response element (RRE), forming a complex that engages with the host nuclear export machinery. To better understand Rev oligomerization, we determined four crystal structures of Rev N-terminal domain dimers, which show that they can pivot about their dyad axis, giving crossing angles of 90° to 140°. In parallel, we performed cryoelectron microscopy of helical Rev filaments. Filaments vary from 11 to 15 nm in width, reflecting variations in dimer crossing angle. These structures contain additional density, indicating that C-terminal domains become partially ordered in the context of filaments. This conformational variability may be exploited in the assembly of RRE/Rev complexes. Our data also revealed a third interface between Revs, which offers an explanation for how the arrangement of Rev subunits adapts to the "A"-shaped architecture of the RRE in export-active complexes.

Bioavailable inhibitors of HIV-1 RNA biogenesis identified through a Rev-based screen.

New antiretroviral agents with alternative mechanisms are needed to complement the combination therapies used to treat HIV-1 infections. Here we report the identification of bioavailable molecules that interfere with the gene expression processes of HIV-1. The compounds were detected by screening a small library of FDA-approved drugs with an assay based on measuring the displacement of Rev, and essential virus-encoded protein, from its high-affinity RNA binding site. The antiretroviral activity of two hits was based on interference with post-integration steps of the HIV-1 cycle. Both hits inhibited RRE-Rev complex formation in vitro, and blocked LTR-dependent gene expression and viral transcription in cellular assays. The best compound altered the splicing pattern of HIV-1 transcripts in a manner consistent with Rev inhibition. This mechanism of action is different from those used by current antiretroviral agents. The screening hits recognized the Rev binding site in the viral RNA, and the best compound did so with substantial selectivity, allowing the identification of a new RNA-binding scaffold. These results may be used for developing novel antiretroviral drugs.

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.

Biophysical Characterization of Nucleophosmin Interactions with Human Immunodeficiency Virus Rev and Herpes Simplex Virus US11.

Nucleophosmin (NPM1, also known as B23, numatrin or NO38) is a pentameric RNA-binding protein with RNA and protein chaperon functions. NPM1 has increasingly emerged as a potential cellular factor that directly associates with viral proteins; however, the significance of these interactions in each case is still not clear. In this study, we have investigated the physical interaction of NPM1 with both human immunodeficiency virus type 1 (HIV-1) Rev and Herpes Simplex virus type 1 (HSV-1) US11, two functionally homologous proteins. Both viral proteins show, in mechanistically different modes, high affinity for a binding site on the N-terminal oligomerization domain of NPM1. Rev, additionally, exhibits low-affinity for the central histone-binding domain of NPM1. We also showed that the proapoptotic cyclic peptide CIGB-300 specifically binds to NPM1 oligomerization domain and blocks its association with Rev and US11. Moreover, HIV-1 virus production was significantly reduced in the cells treated with CIGB-300. Results of this study suggest that targeting NPM1 may represent a useful approach for antiviral intervention.

Thermodynamics of Rev-RNA interactions in HIV-1 Rev-RRE assembly.

The HIV-1 protein Rev facilitates the nuclear export of intron-containing viral mRNAs by recognizing a structured RNA site, the Rev-response-element (RRE), contained in an intron. Rev assembles as a homo-oligomer on the RRE using its α-helical arginine-rich-motif (ARM) for RNA recognition. One unique feature of this assembly is the repeated use of the ARM from individual Rev subunits to contact distinct parts of the RRE in different binding modes. How the individual interactions differ and how they contribute toward forming a functional complex is poorly understood. Here we examine the thermodynamics of Rev-ARM peptide binding to two sites, RRE stem IIB, the high-affinity site that nucleates Rev assembly, and stem IA, a potential intermediate site during assembly, using NMR spectroscopy and isothermal titration calorimetry (ITC). NMR data indicate that the Rev-IIB complex forms a stable interface, whereas the Rev-IA interface is highly dynamic. ITC studies show that both interactions are enthalpy-driven, with binding to IIB being 20-30 fold tighter than to IA. Salt-dependent decreases in affinity were similar at both sites and predominantly enthalpic in nature, reflecting the roles of electrostatic interactions with arginines. However, the two interactions display strikingly different partitioning between enthalpy and entropy components, correlating well with the NMR observations. Our results illustrate how the variation in binding modes to different RRE target sites may influence the stability or order of Rev-RRE assembly and disassembly, and consequently its function.