2-Aminopurine Fluorescence: Discrimination Between Specific and Unspecific Ligand Binding to the Kissing-Loop Dimer of the HIV-1 RNA

Abstract

The fluorescent 2-aminopurine probe (2-AP) incorporated into the loop of 23-mer RNA hairpin of HIV-1 genome dimerization initiation site (DIS) was used for discrimination of specific and unspecific binding of paromomycin and spermine to the kissing loop dimer (KD) formed in solution. While both ligands stabilized the KD RNA structure, only paromomycin binding resulted in significant increase of 2-AP fluorescence. These observations suggest that the 2-AP fluorescent RNA construct might be useful for selecting ligands specifically binding the HIV-1 kissing loop RNA dimer.     

Interaction of the dimeric form of retroviral RNA with paromonycin and magnesium as revealed using 2-aminopurine fluorescence

Studying the dimeric RNA structural organization is a step toward the understanding of retroviral genomic RNA dimerization. A kissing loop dimer is rearranged into an extended dimer during maturation of the virus particle. The extended dimer formation may be inhibited by ligands interacting with the RNA kissing loop dimer. A study was made of the interaction of dimeric RNA with paromomycin and magnesium ions. RNA dimers were formed from two hairpin RNAs having complementary sequences in the loop. The structural features of RNA dimers and the influence of the ligands were inferred from the fluorescence of 2-aminopurine (2-AP) incorporated in one of the two RNA hairpin sequences. As dimeric RNA interacted with paromomycin, 2-AP fluorescence increased. The increase was explained by a flipping of the fluorescent base out of the RNA structure. The binding constants and stoichiometry were estimated for dimeric RNA binding with paromomycin. An RNA dimer was found to interact with two paromomycin molecules; the binding constant was approximately the same (about 3 × 10⁵ M⁻¹) for both types of dimers. It was observed that the antibiotic and Mg²⁺ ions compete for binding to the hairpin RNA dimer and that one paromomycin molecule is displaced by one Mg²⁺ ion.     

Effect of aminoglycoside antibiotics on the conformation of the unpaired loop adenine of avian leucosis virus RNA as revealed using 2-aminopurine fluorescence

The fluorescent properties of 2-aminopurine (2-AP) incorporated in an RNA sequence are used to study the structural dynamics and local changes of the retroviral RNA structure. Using 2-AP, the conformational states of the unpaired loop adenine in avian leucosis virus RNA were studied upon its interaction with aminoglycoside antibiotics. The intensity of 2-AP fluorescence in the monomeric RNA hairpin was higher than in both RNA dimers. The intensity of fluorescence in the extended dimer was significantly lower than in the kissing loop dimer. The finding was be explained by the fact that stacking contacts in the extended dimer produce a more compact loop structure than in the kissing loop dimer. When the binding of aminogycoside antibiotics with the kissing loop dimer RNA was analyzed, only tobramycin increased the intensity of 2-AP fluorescence almost threefold. The results showed that 2-AP fluorescence is suitable for detecting local changes in complexes of retroviral RNA with ligands.     

Dimerization of short RNA fragments from avian leukosis virus as revealed with 2-aminopurine fluorescence

The dimerization of genomic retroviral RNA is well studied for several groups of viruses, the dimerization of human immunodeficiency (HIV) RNA being investigated in more detail. Regions of dimerization apparently involve the short sequences RNA which are directly responsible for the formation of two type dimers: kissing loop-loop (KD) and linear (LD). The 5'-end sequences from RNA avian viruses, where the dimers are basically formed, considerably differ from those of HIV. However, as it was described earlier, the mechanism of dimerization of RNA from human immunodeficiency and from avian leukosis viruses are identical. The fluorescence of adenine analogue 2-aminopurine (2-AP) incorporated into loop sequence of short fragments RNA ALV was used for analysis of dimers formation. Using the temperature dependence of fluorescence intensity 2-AP we have determined RNA melting temperature under various conditions for KD RNA ALV formed by two strands. Effects of magnesium and aminoglycoside antibiotic paromomycin on stabilization of kissing loop-loop dimer RNA have been studied. Under the experimental conditions KD RNA ALV was found to have the stability at the magnesium concentration higher than 1 mM and at paromomycin concentration higher than 2.5 mkM.     

Dimerization of short RNA fragments of avian retroviruses as revealed using 2-aminopurine fluorescence

Dimerization of retroviral RNA is known in detail for several groups of viruses, especially the human immunodeficiency virus (HIV). The dimerization region seems to involve short RNA sequences directly responsible for the formation of dimers of two types, kissing loop-loop (KD) and linear (LD). The 5′-terminal sequences, where dimers are mostly formed, substantially differ between avian retroviruses and HIV, while the mechanisms of their RNA dimerization are the same. RNA dimerization was studied using the adenine analog 2-aminopurine (2-AP), which was incorporated into the loop sequence of short fragments of the avian leucosis virus (ALV) RNA. A temperature dependence of 2-AP fluorescence was used to study the KD formation under various conditions. Magnesium ions and the aminoglycoside antibiotic paromomycin were tested for the effect on KD stability. The highest KD stability was observed at >1 mM Mg²⁺ and >2.5 μM paromomycin.     

Mechanism of nucleocapsid protein catalyzed structural isomerization of the dimerization initiation site of HIV-1

Dimerization of two homologous strands of genomic RNA is an essential feature of retroviral replication. In the human immunodeficiency virus type 1 (HIV-1), a conserved stem-loop sequence, the dimerization initiation site (DIS), has been identified as the domain primarily responsible for initiation of this aspect of viral assembly. The DIS loop contains an autocomplementary hexanucleotide sequence flanked by highly conserved 5' and 3' purines and can form a homodimer through a loop-loop kissing interaction. In a structural rearrangement activated by the HIV-1 nucleocapsid protein (NCp7) and considered to be associated with viral particle maturation, the DIS dimer converts from an intermediate kissing to an extended duplex isoform. Using 2-aminopurine (2-AP) labeled sequences derived from the DIS(Mal) variant and fluorescence methods, the two DIS dimer isoforms have been unambiguously distinguished, allowing a detailed examination of the kinetics of this RNA structural isomerization and a characterization of the role of NCp7 in the reaction. In the presence of divalent cations, the DIS kissing dimer is found to be kinetically trapped and converts to the extended duplex isoform only upon addition of NCp7. NCp7 is demonstrated to act catalytically in inducing the structural isomerization by accelerating the rate of strand exchange between the two hairpin stem helices, without disruption of the loop-loop helix. Observation of an apparent maximum conversion rate for NCp7-activated DIS isomerization, however, requires protein concentrations in excess of the 2:1 stoichiometry estimated for high-affinity NCp7 binding to the DIS kissing dimer, indicating that transient interactions with additional NCp7(s) may be required for catalysis.     

Melting of model HIV-1 stem-loop 1 RNA dimers monitored by 2-aminopurine fluorescence

Viral maturation of HIV-1 involves refolding of its genomic RNA, which is believed to include a rearrangement of the SL1 stem-loop from a metastable conformation called kissing loop dimer (KD) to a stable one termed extended dimer (ED). To investigate this rearrangement in vitro we have studied the thermal melting of the RNA dimers formed by slightly modified 23-nucleotide SL1 RNA of HIV-1 Mal. Local structural changes in the RNA dimers during the melting were monitored by changes in the fluorescence of 2-aminopurine (2AP) incorporated in predetermined positions of RNA. We have shown that the stem regions of both preformed KD and ED melt in the temperature interval from 75 ° C to 90 ° C. Kissing loop interface of the KD RNA is found to be disrupted at lower temperatures from 20 ° C to 55 ° C, at which the stem regions remain intact. Conversion of the preformed KD to ED overcoming the kinetic barrier occurs between 55 ° C and 65 ° C. The melting of "loop-loop" regions in both preformed and newly formed EDs takes place around 70 ° C. Our finding that thermoinduced KD-to-ED conversion is preceded by transient dissociation of loop-loop interface disagrees with a common idea of strand exchange without disruption of loop-loop-contact.     

Evidence that a Kissing Loop Structure Facilitates Genomic RNA Dimerisation in HIV-1

Genomic RNA isolated from retroviral particles is a dimer composed of two identical strands. A region called the dimer linkage signal close to the 5′ end of the RNA may be involved in forming the dimer. Several models for the formation of the HIV-1 RNA dimer have been proposed. In the kissing loop model, dimerisation results from base-pairing between homologous sequences in an RNA stem – loop. In the guanine tetrad model interstrand guanine contacts form the dimer. We have made mutations preventing the dimerisation of subgenomic RNAsin vitroby these mechanisms. To prevent the kissing loop dimer forming we changed the complementary loop sequence from 711GCGCGC716 to 711AAACGC716. To prevent the guanine tetrad dimer forming we changed G819 to U. These mutations were introduced into a clone of HIV-1_NL4-3separately and collectively. All three clones produced infectious virions. Dimeric RNA with similar thermal stabilities was isolated from viruses containing either the single or the double mutations. The results suggest that sequences involved in forming a guanine tetrad are not important for HIV-1 RNA dimerisation. In contrast sequences involved in forming a kissing loop complex are not absolutely required, but are important in forming a stable HIV-1 RNA dimer.     

Nucleocapsid protein-mediated maturation of dimer initiation complex of full-length SL1 stemloop of HIV-1: sequence effects and mechanism of RNA refolding

Specific binding of HIV-1 viral protein NCp7 to a unique 35-base RNA stem-loop SL1 is critical for formation and packaging of the genomic RNA dimer found within HIV-1 virions. NCp7 binding stimulates refolding of SL1 from a metastable kissing dimer (KD) into thermodynamically stable linear dimer (LD). Using UV melting, gel electrophoresis and heteronuclear NMR, we investigated effects of various site-specific mutations within the full-length SL1 on temperature- or NCp7-induced refolding in vitro. Refolding involved intramolecular melting of SL1 stems but not dissociation of the intermolecular KD interface. Refolding required only two NCp7 molecules per KD but was limited by the amount of NCp7 present, implying that the protein does not catalytically promote refolding. Efficient refolding depended strictly on the presence and, to a lesser degree, on sequence of a highly conserved G-rich internal loop that normally limits thermal stability of the SL1 stem. Adding two base pairs to the lower stem created a hyperstable SL1 mutant that failed to refold, even when bound by NCp7at high stoichiometries. NMR analysis of these kinetically trapped mutant RNA–protein complexes indicated that NCp7 initiates refolding by dissociating base pairs in the upper stem of SL1. This study illuminates structural transitions critical for HIV-1 assembly and replication.     

194 Energetics and dynamics of adenine protonation in HIV-1’s dimerization initiation site

HIV-1, like most retroviruses, packages two homologous copies of its RNA genome. The two RNA strands are non-covalently linked near their 5’ends. The proposed dimerisation initiation site is a 35-nucleotide (nt) stem loop capable of forming loop–loop interactions (a kissing dimer) via its highly conserved 6-nt loop palindrome. In a structural transformation affected by temperature, salt concentration, and by the HIV-1 nucleocapsid protein, the initial, kinetically-stable kissing dimer (KD) converts to a thermodynamically-stable extended dimer. It has been suggested that this in vitro observed rearrangement is associated with the in vivo viral genome maturation. Mihailescu et al. demonstrated enhanced rearrangement dynamics triggered by the protonation of a specific adenine residue at the genome sequence location 272 (A272) (Mihailescu, 2004). They suggested that the local environment of A272 caused its N1 atom’s pK_a to shift upwards (more basic) by approximately 2.5 pH units when compared with 5’-adenosine monophosphate (5’-AMP). In this work, we investigated the dynamics and energetics of protonating A272’s N1 atom with explicit solvent molecular dynamics (MD) simulations, Thermodynamic Integration (TI), and the Poisson–Boltzmann equation (PB). Two initial structures were used, an NMR solution structure (PDB ID: 2D19) and an X-ray crystal structure (PDB ID: 1XP7), where A272 was found inside (NMR) and outside (X-ray) the helical axis respectively. MD simulations showed when A272 started,it remained inside the KD’s axis, and when outside, it attempted to insert itself within the axis. Calculated pK_a shifts obtained from solving the PB equation were approximately?+?2.2 and?+?1.0 pH units for the NMR and X-ray structures respectively; while TI calculations performed with the NMR structure yielded a shift of approximately?+?2.5 pH units. Our simulations confirmed the strong influence of A272's local environment on its calculated pK_a when inserted in the helical axis. A272's N1 atom was approximately 200 times more likely protonated when compared with 5'-AMP. Also, protonated A272s were more energetically favoured in the kissing dimer versus an isolated monomer due to a diminished positive electrostatic potential near A272, while in the dimer. Overall, our computational investigations affirm the experimental suggestion that A272 in the kissing dimer is more likely to be protonated at physiological conditions and this protonation may trigger the structural rearrangement of the initial kissing dimer to form the extended dimer.     

The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA

RNA dimerization is an essential step in the retroviral life cycle. Dimerization and encapsidation signals, closely linked in HIV-2, are located in the leader RNA region. The SL1 motif and nucleocapsid protein are considered important for both processes. In this study, we show the structure of the HIV-2 leader RNA (+1-560) captured as a loose dimer. Potential structural rearrangements within the leader RNA were studied. In the loose dimer form, the HIV-2 leader RNA strand exists in vitro as a single global fold. Two kissing loop interfaces within the loose dimer were identified: SL1/SL1 and TAR/TAR. Evidence for these findings is provided by RNA probing using SHAPE, chemical reagents, enzymes, non-denaturing PAGE mobility assays, antisense oligonucleotides hybridization and analysis of an RNA mutant. Both TAR and SL1 as isolated domains are bound by recombinant NCp8 protein with high affinity, contrary to the hairpins downstream of SL1. Foot-printing of the SL1/NCp8 complex indicates that the major binding site maps to the SL1 upper stem. Taken together, these data suggest a model in which TAR hairpin III, the segment of SL1 proximal to the loop and the PAL palindromic sequence play specific roles in the initiation of dimerization.     

Identification of the in vitro HIV-2/SIV RNA dimerization site reveals striking differences with HIV-1

Although their genomes cannot be aligned at the nucleotide level, the HIV-1/SIVcpz and the HIV-2/SIVsm viruses are closely related lentiviruses that contain homologous functional and structural RNA elements in their 5'-untranslated regions. In both groups, the domains containing the trans-activating region, the 5'-copy of the polyadenylation signal, and the primer binding site (PBS) are followed by a short stem-loop (SL1) containing a six-nucleotide self-complementary sequence in the loop, flanked by unpaired purines. In HIV-1, SL1 is involved in the dimerization of the viral RNA, in vitro and in vivo. Here, we tested whether SL1 has the same function in HIV-2 and SIVsm RNA. Surprisingly, we found that SL1 is neither required nor involved in the dimerization of HIV-2 and SIV RNA. We identified the NarI sequence located in the PBS as the main site of HIV-2 RNA dimerization. cis and trans complementation of point mutations indicated that this self-complementary sequence forms symmetrical intermolecular interactions in the RNA dimer and suggested that HIV-2 and SIV RNA dimerization proceeds through a kissing loop mechanism, as previously shown for HIV-1. Furthermore, annealing of tRNA(3)(Lys) to the PBS strongly inhibited in vitro RNA dimerization, indicating that, in vivo, the intermolecular interaction involving the NarI sequence must be dissociated to allow annealing of the primer tRNA.     

The dimer initiation site hairpin mediates dimerization of the human immunodeficiency virus, type 2 RNA genome

The untranslated leader of retroviral RNA genomes encodes multiple structural signals that are critical for virus replication. In the human immunodeficiency virus, type 1 (HIV-1) leader, a hairpin structure with a palindrome-containing loop is termed the dimer initiation site (DIS), because it triggers in vitro RNA dimerization through base pairing of the loop-exposed palindromes (kissing loops). Controversy remains regarding the region responsible for HIV-2 RNA dimerization. Different studies have suggested the involvement of the transactivation region, the primer binding site, and a hairpin structure that is the equivalent of the HIV-1 DIS hairpin. We have performed a detailed mutational analysis of the HIV-2 leader RNA, and we also used antisense oligonucleotides to probe the regions involved in dimerization. Our results unequivocally demonstrate that the DIS hairpin is the main determinant for HIV-2 RNA dimerization. The 6-mer palindrome sequence in the DIS loop is essential for dimer formation. Although the sequence can be replaced by other 6-mer palindromes, motifs that form more than two A/U base pairs do not dimerize efficiently. The inability to form stable kissing-loop complexes precludes formation of dimers with more extended base pairing. Structure probing of the DIS hairpin in the context of the complete HIV-2 leader RNA suggests a 5-base pair elongation of the DIS stem as it is proposed in current RNA secondary structure models. This structure is supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genome dimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.     

Polymorphism of bulged-out residues in HIV-1 RNA DIS kissing complex and structure comparison with solution studies

All retroviruses encapsidate their genome as a dimer of homologous single-stranded RNAs. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) is located in the 5'-untranslated region of the viral genome and consists of a hairpin with a 6 nt self-complementary loop sequence. Genomic RNA dimerization, a crucial step for virion infectivity, is promoted by the formation of a loop-loop complex (or kissing complex) between two DIS hairpins. Crystal structures for the subtypes A, B and F of the HIV-1 DIS kissing complex have now been solved at 2.3 A, 1.9 A and 1.6 A, respectively. They revealed a polymorphism of bulged-out residues showing clearly that their conformation is not a mere consequence of crystal packing. They also provide more insights into ion binding, hydration, and RNA conformation and flexibility. In particular, we observed the binding of spermine to the loop-loop helix, which displaced a magnesium cation important for subtype A DIS dimerization. The excellent agreement between X-ray structures and the results of chemical probing and interference data on larger viral RNA fragments shows that the crystal structures are relevant for the DIS kissing complex present in solution and in viral particles. Accordingly, these structures will be helpful for designing new drugs derived from aminoglycoside antibiotics and targeted against the RNA dimerization step of the viral life-cycle.     

Molecular modeling and dynamics studies of HIV-1 kissing loop structures

Recognition of an RNA loop by another RNA loop is involved in several biological functions. The dimerization of two copies of the HIV-1 genomic RNA is thought to be involved in several steps of the retroviral life cycle. It has been shown that the dimerization of the two HIV-1 RNA genomes is initiated by the so called kissing loop. The 9nt kissing loop consists of a palindromic 6nt sequence that forms Watson-Crick base-pairs at the kissing site in HIV-1. We report the results of our molecular modeling and dynamics studies on two major subtype isolates (MAL and LAI) of HIV-1 kissing loop structures. From our modeling studies, we conclude that the conformation of the loop in the monomer might be closer to the A-RNA-like conformation in order to form an initial kissing structure. This is achieved by the stacking interactions of the bases at the 3' end of the loop and by the intramolecular tertiary interactions of a single linker nucleotide. We discuss the effect of the loop size and the structural limitations on the formation of kissing loop structures. Also, we propose a possible mechanism to convert the kissing loop structure to a stable extended duplex structure without unwinding the stems.     

Structure and stability of RNA/RNA kissing complex: with application to HIV dimerization initiation signal

We develop a statistical mechanical model to predict the structure and folding stability of the RNA/RNA kissing-loop complex. One of the key ingredients of the theory is the conformational entropy for the RNA/RNA kissing complex. We employ the recently developed virtual bond-based RNA folding model (Vfold model) to evaluate the entropy parameters for the different types of kissing loops. A benchmark test against experiments suggests that the entropy calculation is reliable. As an application of the model, we apply the model to investigate the structure and folding thermodynamics for the kissing complex of the HIV-1 dimerization initiation signal. With the physics-based energetic parameters, we compute the free energy landscape for the HIV-1 dimer. From the energy landscape, we identify two minimal free energy structures, which correspond to the kissing-loop dimer and the extended-duplex dimer, respectively. The results support the two-step dimerization process for the HIV-1 replication cycle. Furthermore, based on the Vfold model and energy minimization, the theory can predict the native structure as well as the local minima in the free energy landscape. The root-mean-square deviations (RMSDs) for the predicted kissing-loop dimer and extended-duplex dimer are ∼3.0 Å. The method developed here provides a new method to study the RNA/RNA kissing complex.     

Ligand-induced changes in 2-aminopurine fluorescence as a probe for small molecule binding to HIV-1 TAR RNA

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated in part through an interaction between the virally encoded trans-activator protein Tat and the trans-activator responsive region (TAR) of the viral RNA genome. Because TAR is highly conserved and its interaction with Tat is required for efficient viral replication, it has received much attention as an antiviral drug target. Here, we report a 2-aminopurine (2-AP) fluorescence-based assay for evaluating potential TAR inhibitors. Through selective incorporation of 2-AP within the bulge (C23 or U24) of a truncated form of the TAR sequence (delta TAR-ap23 and delta TAR-ap24), binding of argininamide, a 24-residue arginine-rich peptide derived from Tat, and Neomycin has been characterized using steady-state fluorescence. Binding of argininamide to the 2-AP deltaTAR constructs results in a four- to 11-fold increase in fluorescence intensity, thus providing a sensitive reporter of that interaction (KD approximately 1 mM). Similarly, binding of the Tat peptide results in an initial 14-fold increase in fluorescence (KD approximately 25 nM), but is then followed by a slight decrease that is attributed to an additional, lower-affinity association(s). Using the deltaTAR-ap23 and TAR-ap24 constructs, two classes of Neomycin binding sites are detected; the first molecule of antibiotic binds as a noncompetitive inhibitor of Tat/argininamide (KD approximately 200 nM), whereas the second, more weakly bound molecule(s) becomes associated in a presumably nonspecific manner (KD approximately 4 microM). Taken together, the results demonstrate that the 2-AP fluorescence-detected binding assays provide accurate and general methods for quantitatively assessing TAR interactions.     

Stability and conformation of the dimeric HIV-1 genomic RNA 5′UTR

During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5′ untranslated region (5′UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5′UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation. Two 5′UTR dimer conformations, the kissing dimer (KD) and the extended dimer (ED), have been identified in vitro, which differ in the extent of intermolecular basepairing. Whether 5′UTRs from different HIV-1 strains with distinct sequences have access to the same dimer conformations has not been determined. Here, we applied fluorescence cross-correlation spectroscopy and single-molecule Förster resonance energy transfer imaging to demonstrate that 5′UTRs from two different HIV-1 subtypes form (KDs) with divergent stabilities. We further show that both 5′UTRs convert to a stable dimer in the presence of the viral NC protein, adopting a conformation consistent with extensive intermolecular contacts. These results support a unified model in which the genomes of diverse HIV-1 strains adopt an ED conformation.